Selective glucagon-like-peptide-2 (glp-2) analogues

ABSTRACT

GLP-2 analogues are disclosed which comprise one of more substitutions as compared to h[Gly2]GLP-2 and which may have the property of an increased small intestine/colon and stomach/colon selectivity. More particularly, preferred GLP-2 analogues disclosed herein comprise substitutions at one or more of positions (11, 16, 20, 24) and/or (28) of the wild-type GLP-2 sequence, optionally in combination with further substitutions at position (2) and one or more of positions (3, 5, 7), and (10), and/or a deletion of one or more of amino acids (31) to (33) and/or the addition of a N-terminal or C-terminal stabilizing peptide sequence. The analogues are particularly useful for the prophylaxis or treatment of stomach and bowel-related disorders and for ameliorating side effects of chemotherapy.

FIELD OF THE INVENTION

The present invention relates to selective glucagon-like-peptide-2(GLP-2) analogues and their medical use, for example in the prophylaxisor treatment of stomach and bowel-related disorders and for amelioratingthe gastro-intestinal associated side effects of chemotherapy and/orradiation therapy.

BACKGROUND OF THE INVENTION

GLP-2 is a 33-amino-acid peptide derived from specific posttranslationalprocessing of proglucagon in the enteroendocrine L cells of theintestine and in specific regions of the brainstem. It is co-secretedtogether with glucagon-like peptide 1 (GLP-1), oxyntomodulin andglicentin, in response to nutrient ingestion.

GLP-2 induces significant growth of the small intestinal mucosalepithelium via the stimulation of stem cell proliferation in the cryptsand inhibition of apoptosis on the villi (Drucker et al. Proc Natl AcadSci USA. 1996, 93:7911-6). GLP-2 also has growth effects on the colon.GLP-2 also inhibits gastric emptying and gastric acid secretion(Wojdemann et al. J Clin Endocrinol Metab. 1999, 84:2513-7), enhancesintestinal barrier function (Benjamin et al. Gut. 2000, 47:112-9.),stimulates intestinal hexose transport via the upregulation of glucosetransporters (Cheeseman, Am J Physiol. 1997, R1965-71.), and increasesintestinal blood flow (Guan et al. Gastroenterology. 2003, 125, 136-47).

GLP-2 binds to a single G protein-coupled receptor belonging to theclass II glucagon secretin family. The GLP-2 receptor is expressed inthe small intestine, colon and stomach, sites that are known to beresponsive to GLP-2 (Yusta et al. Gastroenterology. 2000, 119: 744-55).However, the target cell for GLP-2 receptor stimulation in thegastrointestinal tract remains unclear and the downstream intracellularmediators coupled to the GLP-2 receptor are poorly understood.

The demonstrated specific and beneficial effects of GLP-2 in the smallintestine have raised much interest as to the use of GLP-2 in thetreatment of intestinal disease or injury (Sinclair and Drucker,Physiology 2005: 357-65). Furthermore GLP-2 has been shown to prevent orreduce mucosal epithelial damage in a wide number of preclinical modelsof gut injury, including chemotherapy-induced enteritis,ischemia-reperfusion injury, dextran sulfate-induced colitis and geneticmodels of inflammatory bowel disease (Sinclair and Drucker Physiology2005: 357-65).

Further, the expression of the GLP-2R mRNA in the stomach, (Yusta etal., 2000) together with the observation that GLP-2 reduces gastricmotility and gastric acid secretion (Meier et al., 2006) provides ampleevidence that the stomach is either directly or indirectly responsive toGLP-2.

Nevertheless the use of GLP-2 or analogues of GLP-2 in conditionscharacterised by damage to the gastric lining has not yet beingexplored.

GLP-2 is secreted as a 33 amino acid peptide with the following sequenceH-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-OH.It is rapidly cleaved at the Alanine (A) in position 2 of the NH₂terminus to the inactive human GLP-2 (3-33) by the enzyme DPP IV. Thisrapid enzymatic degradation of GLP-2(1-33), in addition to renalclearance result in a half life of about 7 minutes for the peptide(Tavares et al., Am. J. Physiol. Endocrinol. Metab. 278:E134-E139,2000).

In U.S. Pat. No. 5,994,500 (Drucker et al.) describes antagonists ofGLP-2 and their effects on the growth of gastrointestinal tissue. It issuggested that the antagonists are formulated as pharmaceuticals to beused in the treatment of hyperplasia or to induce hypoplasia. In U.S.Pat. No. 5,994,500 the structure of mammalian GLP-2 has been altered bymutations, such as substitutions and deletions.

U.S. Pat. No. 6,184,208; U.S. Pat. No. 5,789,379 and U.S. Pat. No.6,184,201 disclose GLP-2 analogues and their medical uses. Theseanalogues are all obtained by substitutions and/or deletions of humanGLP-2.

DaCambra et al. (Biochemistry 2000, 39, 8888-8894) describe thestructural determinants for activity of GLP-2. Examples of suchdeterminants are Phe6 and Thr5, which are referred to as crucial forGLP-2 receptor binding and activation.

In WO 97/39031 the GLP-2 analogue, [Gly2]GLP-2 is disclosed. Here thealanine in position 2 has been replaced with glycine to make the peptideresistant to DPP IV cleavage. The replacement of alanine is shown toincrease the stability and potency of the peptide. The patentapplication describes how the GLP-2 analogue may be used againstdiseases associated with inflammation and destruction of the intestinalepithelial mucosa. These include massive small intestine resection,inflammatory bowel disease, chemotherapy and/or radiation inducedenteritis and ischemic injury.

WO 02/066511 describes GLP-2 analogues having an extended half-life invivo and their use as medicaments in the treatment of gastrointestinaldisorders, such as inflammatory bowel diseases.

WO 01/41779 describes the use of h[Gly2]GLP-2 as a pretreatment forinhibiting chemotherapy induced apoptosis and promoting small intestinalepithelial cell survival.

All references cited herein are expressly incorporated by reference intheir entirety.

The use of GLP-2 or analogues of GLP-2 in the treatment of variousdiseases has been proposed by many scientists. However, there is still aneed for improved and small intestine selective GLP-2 analogues.

SUMMARY OF THE INVENTION

Broadly, the present invention concerns GLP-2 analogues which compriseone or more substitutions as compared to wild-type GLP-2 and which mayhave the property of an increased small intestine versus colonselectivity, an improved biological activity in vivo and/or improvedchemical stability, e.g. as assessed in in vitro stability assays. Moreparticularly, preferred GLP-2 analogues of the present inventioncomprise non-conservative substitutions at one or more of positions 8,11, 12, 13, 16, 17, 18, 20, 21, 24 and/or 28 of the wild-type GLP-2sequence, optionally in combination with further conservative ornon-conservative substitutions at position 2 (as mentioned in theintroduction) and one or more of positions 3, 5, 7 and 10, and/or asubstitution or deletion of one or more of amino acids corresponding topositions 31 to 33 of the wild-type GLP-2 sequence and, optionally, theaddition of an N-terminal or C-terminal stabilizing peptide sequence. Inaddition the GLP-2 analogues of the invention may comprise conservativesubstitutions of one or more amino acids corresponding to positions 9,14, and 15. As well as providing GLP-2 analogues that may have improvedchemical stability and/or biological activity, the present inventionalso relates to providing compounds that have preferential intestinalgrowth promoting activity in the small intestine compared to the colonand vice versa, in particular by including modification at one or moreof positions Asn11 and/or Asn16 and/or Arg20 and/or Asn24 and/or Gln28of wild-type GLP-2.

Accordingly, in one aspect, the present invention provides a GLP-2analogue which is represented by the general Formula I:

R¹—Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²

wherein:

R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl ortrifluoroacetyl;

X2 is Gly, Ala or Sar;

X3 is Glu or Asp;

X5 is Ser or Thr;

X6 is Phe or Pro or a conservative substitution;

X7 is Ser or Thr;

X8 is Asp or Ser or a conservative substitution;

X9 is Glu or Asp or a conservative substitution;

X10 is Met, Leu, Nle or an oxidatively stable Met-replacement aminoacid;

X11 is Y1;

X12 is Thr or Lys or a conservative substitution;

X13 is Ile, Glu or Gln or a conservative substitution;

X14 is Leu, Met or Nle or a conservative substitution;

X15 is Asp or Glu or a conservative substitution;

X16 is Y2;

X17 is Leu or Glu or a conservative substitution;

X18 is Ala or Aib or a non-conservative substitution;

X19 is Ala or Thr or a conservative substitution;

X20 is Y3

X21 is Asp or Ile or a conservative substitution;

X24 is Y4;

X28 is Y5;

X31 is Pro, Ile or deleted;

X32 is Thr or deleted;

X33 is Asp, Asn or deleted;

R² is NH₂ or OH;

wherein

Z¹ and Z² are independently absent or a peptide sequence of 1-10 aminoacid units selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn; and thehydrophaticity profile (HPP) of the residues X11, X16, X20, X24, X28 offormula I calculated as

HPP=Σ hpi_(X11)+hpi_(X16)+hpi_(X20)+hpi_(X24)+hpi_(X28) is ≧−10

wherein

Y₁, Y₂, Y₄, and Y₅ can individually be selected from the groupconsisting of Asn, Asp, Glu, Gln, Lys, His, Arg, Ala, Ser, Thr, Pro,Gly, Leu, Ile, Val, Met or Phe; and

Y₃ can be selected from the group consisting of Asn, Asp, Glu, Gln, His,Arg, Ala, Ser, Thr, Pro, Gly, Leu, Ile, Val, Met or Phe;

with the proviso that when X20 is Arg then X11 is Ser, X16 is Ala, X24is Ala, X28 is Ala and Z2 is Lys, or a pharmaceutically acceptable saltor derivative thereof.

In one embodiment the invention comprises a glucagon-like peptide 2(GLP-2) analogue as described above wherein HPP is ≧−4

In another embodiment the invention comprises a glucagon-like peptide 2(GLP-2) analogue as described above wherein HPP≧0

In another aspect, the present invention provides a GLP-2 analogue whichis represented by general Formula II:

R¹—Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²

wherein:

R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl ortrifluoroacetyl

X2 is Gly, Ala or Sar

X3 is Glu or Asp

X5 is Ser or Thr

X6 is Phe or Pro

X7 is Ser or Thr

X8 is Asp or Ser

X9 is Glu or Asp

X10 is Met, Leu, Nle or an oxidatively stable Met-replacement amino acid

X11 is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val

X12 is Thr or Lys

X13 is Ile, Glu or Gln

X14 is Leu, Met or Nle

X15 is Asp or Glu

X16 is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val

X17 is Leu or Glu

X18 is Ala or Aib

X19 is Ala or Thr

X20 is Asn, Arg, Ala, Glu, Gly, Ile, Leu, Met, Phe Ser, Thr or Val

X21 is Asp or Ile

X24 is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val

X28 is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val

X31 is Pro, Ile or deleted

X32 is Thr or deleted

X33 is Asp, Asn or deleted

R² is NH₂ or OH;

Z¹ and Z² are independently absent or a peptide sequence of 1-10 aminoacid units selected from the group consisting of Ala, Leu, Ser, Thr,Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn; or apharmaceutically acceptable salt or derivative thereof;

with the proviso that when X20 is Arg then X11 is Ser, X16 is Ala, X24is Ala, X28 is Ala and Z2 is Lys.

In one embodiment the invention comprises a GLP-2 analogue as describedabove wherein

X11 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val

X16 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val

X20 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val

X24 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val

X28 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val.

In another embodiment the invention comprises a GLP-2 analogue asdescribed above wherein

X11 is Ala, Ile, Leu, Phe or Val;

X16 is Ala, Ile, Leu, Phe or Val

X20 is Ala, Ile, Leu, Phe or Val

X24 is Ala, Ile, Leu, Phe or Val

X28 is Ala, Ile, Leu, Phe or Val.

In yet another embodiment the invention comprises a GLP-2 analogue asdescribed above wherein the GLP-2 analogue has at least 60% amino acidsequence identity to wild-type GLP-2 (1-33) and has the biologicalactivity of causing an increase in intestinal mass in vivo.

In a further embodiment the invention comprises an GLP-2 analogue withmore than one of the substitutions (i.e. more than substitution relativeto the wild type sequence given above) at positions X11, X16, X20, X24and/or X28 and/or one of more of said substitutions in combination withone or more substitutions at positions X3, X5, X7 and/or X10.

In still another embodiment the invention comprises an GLP-2 analoguewith substitutions at position X10 is Leu, Nle, or an oxidatively stableMet-replacement amino acid, such as Met(O) or Met(O)₂. Thus,additionally or alternatively to the substitutions already mentioned,the GLP-2 analogue may have Leu, Nle, or an oxidatively stableMet-replacement amino acid, such as Met(O) or Met(O)₂ at position X10.

In some embodiments of the present invention, the GLP-2 analogue has atleast 60% amino acid sequence identity to wild-type GLP-2 (1-33) havingthe sequence set out in the background of the application, morepreferably at least 63% sequence identity, more preferably at least 66%sequence identity and still more preferably at least 69% sequenceidentity.

“Percent (%) amino acid sequence identity” with respect to the GLP-2polypeptide sequences is defined as the percentage of amino acidresidues in a candidate sequence that are identical with the amino acidresidues in the wild-type GLP-2 sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity, and not considering any conservative substitutions aspart of the sequence identity. Sequence alignment can be carried out bythe skilled person using techniques well known in the art for exampleusing publicly available software such as BLAST, BLAST2 or Alignsoftware, see:

Altschul et al (Methods in Enzymology, 266:460-480 (1996);

http://blast.wustl/edu/blast/README.html) or Pearson et al (Genomics,46, 24,36, 1997) and http://molbiol.soton.ac.uk/compute/align.html forthe Align program.

The percentage sequence identities used herein and in accordance withthe present invention are determined using these programe with theirdefault settings. More generally, the skilled person can readilydetermine appropriate parameters for determining alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared.

In another embodiment of the present invention the GLP-2 analogue asdescribed above comprises more than one of the substitutions (i.e. morethan one substitution relative to the wild type sequence given above) atpositions X3, X5, X7, X11, X16, X20, X24, X28, X31, X32 and/or X33.

In still another embodiment of the present invention the GLP-2 analogueas described above comprises one or more of substitutions selected fromX11, X16, X20, X24, X28 is Ile, Ala, Leu, Phe or Val; and the amino acidresidues in positions X31, X32 and X33 are optionally deleted; or apharmaceutically acceptable salt or derivative thereof.

In still another embodiment of the present invention the GLP-2 analogueas described above comprises a substitution (i.e. a substitutionrelative to the wild type sequence given above) at one or more ofpositions X11, X16, X20, X24 and/or X28.

In a preferred embodiment of the present invention the GLP-2 analogue asdescribed above is disclosed in Table 1 or Table 2 herein or apharmaceutically acceptable salt or derivative thereof.

In a preferred embodiment of the present invention the GLP-2 analogue isdefined by the general formula III:

R1-His-Gly-Glu-Gly-Ser-Phe-Ser-X8-Glu-Leu-X11-Thr-Ile-Leu-X15-X16-Leu-Ala-Ala-X20-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-Ile-Thr-Asp-NH_(2;)

wherein

R¹ is hydrogen, C₁₋₄ alkyl (e.g. methyl), acetyl, formyl, benzoyl ortrifluoroacetyl

X8 is Asp or Ser, preferably Asp;

X11 is Ser, Ala, Glu, Lys or Asn;

X15 is Glu or Asp, preferably Glu;

X16 is Ser, Ala or Glu;

X20 is Ser, Ala, or Glu;

X24 is Ser, Ala or Glu; and

X28 is Ser, Ala, Gln or Glu;

or a pharmaceutically acceptable salt or derivative thereof.

Interesting compounds of the invention (formula I) are described in thefollowing table, where said compounds may be modified at the N-terminusas described for R1 in formula III and including a pharmaceuticallyacceptable salt or derivative thereof:

ZP2263His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ser-Thr-Ile-Leu-Glu-Ser-Leu-Ala-Ala-Ser-Asp-Phe-Ile-Ser-Trp-Leu-Ile-Ser-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2264His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ala-Leu-Ala-Ala-Ala-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2266His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Glu-Thr-Ile-Leu-Glu-Glu-Leu-Ala-Ala-Glu-Asp-Phe-Ile-Glu-Trp-Leu-Ile-Glu-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2267His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ser-Thr-Ile-Leu-Glu-Ser-Leu-Ala-Ala-Ala-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2268His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ala-Leu-Ala-Ala-Ser-Asp-Phe-Ile-Ser-Trp-Leu-Ile-Ser-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2269His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Lys-Thr-Ile-Leu-Glu-Ser-Leu-Ala-Ala-Ala-Asp-Phe-Ile-Glu-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2270His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-LeuN-Thr-Ile-Leu-Glu-Ser-Leu-Ala-Ala-Ser-Asp-Phe-Ile-Ser-Trp-Leu-Ile-Ser-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2272His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ser-Leu-Ala-Ala-Ala-Asp-Phe-Ile-Ser-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂ ZP2242His-Gly-Glu-Gly-Ser-Phe-Ser-Ser-Glu-Leu-Ser-Thr-Ile-Leu-Asp-Ala-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-OH

Further compounds of the invention are shown in Table 1, below.

The present invention provides compounds that have preferential growthpromoting activity in the small intestine compared to the colon. Inparticular, the experiments described herein show that certainsubstitutions at positions X11 and/or X16 and/or Asn20 and/or X24 and/orX28 of wild-type GLP-2 provide a preferential increase of the smallintestine weight when administered to test animals compared to theincrease in colon mass. These findings mean that the exemplifiedcompounds may be useful for treating conditions where it is advantageousto have an increased growth promoting effect in the small intestine,while having a lower effect on the colon or in conditions where smallintestine is damaged and the colon intact.

Thus, compounds that are preferred for causing growth of the smallintestine typically comprise one or more substitutions (i.e. relative tothe wild type sequence given above) at positions 11, 16, 20, 24 and/orX28 of wild-type GLP-2. Such compounds may selectively cause growth ofthe small intestine rather than the colon. They may therefore be usedfor conditions affecting or related to the small intestine.

Preferably, such small intestine-selective compounds comprisesubstitutions (i.e. relative to the wild type sequence given above) atmore than one of positions X11, X16, X20, X24, and/or X28. Thus thesmall-intestine-selective compounds may comprise more than one of thesubstitutions where X11 is Ser, X16 is Ala, X20, X24 is Ala, X28 is Ala,X31 is Ile, X32 is Thr and X33 is Asp. The amino acid residues inpositions X31, X32 and X33 may optionally be deleted.

In small intestine-selective compounds, each of X11, X16, X20, X24 andX28 may independently be Ala, Ser, Gly or Thr.

For example, each of X11 and X16 may independently be Ala or Ser, andX20, X24 and X28 may independently be Ala, Ser, Gly or Thr.

For example, X11 and X16 may both be Ala, and X20, X24 and X28 mayindependently be Ala, Ser, Gly or Thr.

For example, X11 and X16 may both be Ala, and X20, X24 and X28 may allindependently be Ala or Ser.

It will be appreciated that other residue combinations falling outsidethese general criteria may also provide small intestine selectivity.

Preferred combinations of residues at positions X11, X16, X20, X24 andX28 include Ser/Ser/Ser/Ser/Ser; Ala/Ala/Ser/Ser/Ser,Ala/Ala/Ala/Ala/Ser, Ser/Ala/Ser/Ser/Ser, Ala/Ser/Ser/Ser/Ser;Ala/Ala/Ala/Ser/Ala; Ala/Ala/Ser/Ala/Ala; Ser/Ala/Ala/Ala/Ala;Ser/Ala/Arg/Ala/Ala; Ala/Ser/Ala/Ser/Ala; Ala/Ala/Ala/Ala/Ala.

Exemplified compounds preferentially stimulating epithelial growth inthe small intestine include ZP2264, ZP2268, ZP2242, ZP2272, ZP2411,ZP2380, ZP2384, ZP2398, ZP2417, ZP2423, ZP2385, ZP2399, ZP2418, ZP2381,ZP2420 and ZP2397.

The invention also extends to compounds having preferential growthpromoting activity in the stomach compared to colon. They may thereforebe used for conditions affecting or related to the stomach.

Preferably, such stomach-selective compounds comprise substitutions(i.e. relative to the wild type sequence given above) at more than oneof positions X11, X16, X20, X24, and/or X28. The amino acid residues inpositions X31, X32 and X33 may optionally be deleted.

For example, in stomach-selective compounds,

X11 may be Leu, Phe or Lys.

X16 may be Leu, Phe or Lys.

X20 may be Ala or Ser.

X24 may be Ala, Ser or Lys.

X28 may be Ala, Ser or Lys.

For example, X11 and X16 may independently be Leu or Phe, X24 and X28may independently be Lys or Ser, and X20 may be Ser.

For example, X11 and X16 may independently be Leu or Phe, and X20, X24and X28 may be Ser.

It will be appreciated that other residue combinations falling outsidethese general criteria may also provide stomach selectivity.

Preferred combinations of residues at positions X11, X16, X20, X24 andX28 include Lys/Lys/Lys/Lys/Lys; Phe/Phe/Ser/Ser/Ser;Leu/Leu/Ala/Ala/Ala; and Leu/Leu/Ser/Ser/Ser.

Exemplified compounds preferentially stimulating epithelial growth inthe stomach include ZP2400, ZP2412, ZP2396, ZP2395, ZP2394 and ZP2401.

Preferably, the GLP-2 analogue maintains an observed purity of at least70% relative to the initial purity in at least one of the degradationtests described in Example 7 below. Additionally or alternatively, itmay maintain an observed purity of at least 60% relative to initialpurity in a solution of HCl 0.1 M after 12 days. Additionally oralternatively it may maintain an observed purity of at least 70%relative to initial purity in a solution of NH4HCO3 0.1 M after 6 days.

Another aspect of the present invention relates to GLP-2 analogues asdescribed above having at least an EC50 of 1 nM and thus are defined asGLP-2 agonists. The present compounds were analysed as described inexample 9. In FIG. 5 the EC50 of ZP2264 is shown as an example of anGLP-2 agonist.

In a further aspect, the present invention provides a compositioncomprising a GLP-2 analogue as defined herein, or a salt or derivativethereof, in admixture with a carrier. In preferred embodiments, thecomposition is a pharmaceutically acceptable composition and the carrieris a pharmaceutically acceptable carrier. The GLP-2 peptide analogue maybe a pharmaceutically acceptable acid addition salt of the GLP-2analogue.

In one embodiment the invention relates to the use of a GLP-2 analogueas defined herein as a pharmaceutical composition, which is formulatedas a liquid suitable for administration by injection or infusion, orwhich is formulated to cause slow release of said GLP-2 analogue.

In a further aspect, the present invention provides a GLP-2 analogue asdefined herein, or a salt thereof, for use in therapy.

The present invention further provides the use of a GLP-2 analogue asdescribed above for the preparation of a medicament for the treatmentand/or prevention of a stomach and bowel-related disorder.

In a preferred embodiment the present invention relates to the use of aGLP-2 analogue, or a salt or derivative thereof for the preparation of amedicament for the treatment and/or prevention of stomach andbowel-related disorders, such as the treatment of neonatals withcompromised intestine function, osteoporosis, and DPP-IV(dipeptidylpeptidase-IV) mediated conditions. By way of example, thestomach and bowel-related disorders include ulcers, Zollinger-Ellisonsyndrome, gastritis, digestion disorders, malabsorption syndromes,short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease(Crohns disease and Ulcerative colitis), celiac sprue (for examplearising from gluten induced enteropathy or celiac disease), tropicalsprue, hypogammaglobulinemic sprue, enteritis, irritable bowel syndromeassociated with diarrhea, small intestine damage and short bowelsyndrome.

in another preferred embodiment the present invention relates to the useof a GLP-2 analogue as described above wherein said stomach andbowel-related disorder is radiation enteritis, infectious orpost-infectious enteritis, or small intestinal damage due to toxic orother chemotherapeutic agents.

The present invention further provides the use of a GLP-2 analogue asdefined herein for the preparation of a medicament for the treatmentand/or prevention of a side effect of chemotherapy or radiationtreatment.

Said side effect of chemotherapy and/or radiation is diarrhea, abdominalcramping, vomiting or structural and functional damage of the intestinalepithelium resulting from chemotherapy treatment.

The present invention further provides the use of a GLP-2 analogue asdefined herein for the preparation of a medicament for the treatment ofneo-natals, osteoporosis or DPP-IV (dipeptidylpeptidase-IV) mediatedconditions.

The invention also provides a therapeutic kit comprising a cancerchemotherapy drug and a GLP-2 analogue of the present invention, eachoptionally in combination with a pharmaceutically acceptable carrier.The two therapeutic agents may be packaged separately (e.g. in separatevials) for separate administration, or may be provided in the samecomposition. Thus the invention further provides a pharmaceuticalcomposition comprising a cancer chemotherapy drug and a GLP-2 analogueof the present invention in combination with a pharmaceuticallyacceptable carrier.

For patients having gastrointestinal mucosal neoplasia, or an increasedrisk of gastrointestinal mucosal neoplasia, it may be desirable toselect a compound so as to reduce or abrogate the risk of reduced sideeffects such as stimulation or aggravation of gastrointestinal mucosalneoplasia. For example, when selecting a compound for treating a patientwith colon neoplasia (whether benign or malignant), or at risk ofdeveloping colon neoplasia, it may be more appropriate to select acompound which is selective for the small intestine over the colon thana non-selective compound or a compound which is selective for the colonover the small intestine.

In other aspects, the present invention provides the use of the GLP-2analogues for the preparation of a medicament for the treatment and/orprevention of malnutrition, for example conditions such as the wastingsyndrome cachexia and anorexia.

In a further aspect, the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding a GLP-2 analogue asdefined herein.

In further aspects, the present invention provides an expression vectorcomprising the above nucleic acid sequence, optionally in combinationwith sequences to direct its expression, and host cells transformed withthe expression vectors. Preferably the host cells are capable ofexpressing and secreting the GLP-2 analogue. In a still further aspect,the present invention provides a method of producing the GLP-2 analogue,the method comprising culturing the host cells under conditions suitablefor expressing the GLP-2 analogue and purifying the GLP-2 analogue thusproduced.

The invention further provides a nucleic acid of the invention, anexpression vector of the invention, or a host cell capable of expressingand secreting a GLP-2 analogue of the invention, for use in therapy. Itwill be understood that the nucleic acid, expression vector and hostcells may be used for treatment of any of the disorders described hereinwhich may be treated with the GLP-2 analogues themselves. References toa therapeutic composition comprising a GLP-2 analogue of the invention,or administration of a GLP-2 analogue of the invention, should thereforebe construed to encompass administration of a nucleic acid, expressionvector or host cell of the invention except where the context demandsotherwise.

In one embodiment the present invention relates to the use of a nucleicacid molecule, an expression vector, or a host cell as defined herein,in the preparation of a medicament for the treatment and/or preventionof a stomach and bowel-related disorder, or for the treatment and/orprevention of a side effect of chemotherapy or radiation treatment, orfor the treatment of neo-natals, osteoporosis or DPP-IV(dipeptidylpeptidase-IV) mediated conditions.

In a further aspect, the present invention provides a method of treatinga stomach and bowel-related disorder in a patient in need thereof byadministering an effective amount a nucleic acid, expression vector orhost cell of the invention. Examples of stomach and bowel-relateddisorders are provided above.

In a further aspect, the present invention provides a method of treatingor preventing a side effect of chemotherapy or radiation therapy in apatient in need thereof, the method comprising administering aneffective amount a nucleic acid, expression vector or host cell of theinvention.

In a further aspect, the present invention provides a method of treatingor preventing malnutrition, for example conditions such as the wastingsyndrome cachexia and anorexia, in a patient in need thereof, the methodcomprising administering an effective amount a nucleic acid, expressionvector or host cell of the invention.

Embodiments of the present invention will now be described in moredetail by way of examples and not limitation with reference to theaccompanying figures.

DESCRIPTION OF THE FIGURES

FIG. 1. Change in relative small intestinal mass, vs. vehicle controlsfollowing administration of the reference compound, [Gly2]GLP-2 and thecompounds ZP2264, ZP2266-ZP2268 (800 nmol/kg, once daily for 3 days).Relative small intestinal mass is shown as 100% in the vehicle animals.*:P<0.05, vs. veh, $:P<0.05 vs. [Gly2]GLP-2 treated

FIG. 2. Change in relative small intestinal mass, vs. vehicle controlsfollowing administration of the reference compound, [Gly2]GLP-2 and thecompounds ZP2242, ZP2269 and ZP2272 and (800 nmol/kg, once daily for 3days). Relative small intestinal mass is shown as 100% in the vehicleanimals. *:P<0.05, vs. veh, $:P<0.05 vs. [Gly2]GLP-2 treated

FIG. 3. Change in the ratio of small intestine mass/colon mass, vs.[Gly2]GLP-2-treated animals following administration of the compoundsZP2264, ZP2266-ZP2268 (800 nmol/kg, once daily for 3 days). Relativesmall intestinal (SI) mass is shown as 100% in the vehicle animals.$:P<0.05 vs. [Gly2]GLP-2 treated

FIG. 4. Change in the ratio of small intestine mass/colon mass, vs.[Gly2]GLP-2-treated animals following administration of the compoundsZP2242, ZP2269, ZP2272 and ZP2271 (800 nmol/kg, once daily for 3 days).Relative small intestinal (SI) mass is shown as 100% in the[Gly2]GLP-2-treated animals.

FIG. 5. In vitro screening of ZP2264 for agonism on the GLP-2 receptorrecombinantly expressed in BHK-21 cells. The IC 50 of ZP2264 was3.55E-12, well within the range defined for agonists.

FIG. 6. Small intestine/colon sensitivity index of ZP compoundsselective for the small intestine.

FIG. 7. Stomach/colon sensitivity index of ZP compounds selective forthe stomach.

FIG. 8. Small intestine/colon sensitivity index of unselective ZPcompounds.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless specified otherwise, the following definitions are provided forspecific terms, which are used in the above written description.

Throughout the description and claims the conventional one-letter andthree-letter codes for natural amino acids are used as well as generallyaccepted three letter codes for other α-amino acids, such as sarcosin(Sar), norleucine (Nle) and α-aminoisobutyric acid (Aib). All amino acidresidues in peptides of the invention are preferably of theL-configuration. However, D-configuration amino acids may also bepresent.

As used herein “conservative substitution” means that an amino acidresidue belonging to a certain position of the native human GLP-2peptide sequence has been exchanged with an amino acid residue belongingto the same group (I, II, III, IV, V, 1, 2, 3) as defined in thefollowing table:

I II III IV V A N H M F S D R L Y T E K I W P Q V G C 1 2 3 A G H V S RL T K I C D P Y E F N W Q M

A “non-conservative” substitution as used herein means any othersubstitution of an amino acid residue of the native human GLP-2sequence, e.g. such as substituting with a non-protein amino acid (Sar,Nle, Aib) or substituting with an amino acid which does not belong tothe same group.

To describe the resulting hydrophophatic profile of one side of analpha-helix we have chosen the Hydropathy index's (hpi_(X)) for theindividual amino acids described by Kyte and Doolittle, J. Mol. Biol.(1982) 157, 105-132. In the present invention the helix of interest isoutlined by the amino acid positions X11-X16-X20-X24-X28.

The Hydropathy Index (HPI) of the individual amino acids is describedusing the hydropathy index HPI introduced and defined by Kyte andDoolittle, J. Mol. Biol. (1982) 157, 105-132.

HPI values for the individual amino acids are:

Amino Acid hpi-index I 4.5 V 4.2 L 3.8 F 2.8 C 2.5 M 1.9 A 1.8 G −0.4 T−0.7 W −0.9 S −0.8 Y −1.3 P −1.6 H −3.2 E −3.5 Q −3.5 D −3.5 N −3.5 K−3.9 R −4.5

The resulting hydrophaticity profile (HPP) of the residues X11, X16,X20, X24, X28 of formula I calculated as HPP=Σhpi_(X11)+hpi_(X16)+hpi_(X20)+hpi_(X24)+hpi_(X28)

HPP≧−10

HPP≧−4

HPP≧0

Y₁, Y₂, Y₄, and Y₅ can individually be selected from the group: Asn,Asp, Glu, Gln, Lys, His, Arg, Ala, Ser, Thr, Pro, Gly, Leu, Ile, Val,Met or Phe.

Y₃ can be selected from the group: Asn, Asp, Glu, Gln, His, Arg, Ala,Ser, Thr, Pro, Gly, Leu, Ile, Val, Met or Phe.

For a small intestine-selective compound, within the overall criteriathat HPP≧−10, −4 or 0, it may be desirable that HPI for positions 11 and16 should independently be −0.8≦HPI≦3.8, for example −0.8≦HPI≦2.8, suchas HPI=1.8.

For positions 20, 24 and 28 it may be desirable that HPI shouldindependently be −0.8≦HPI_(20,24,28)≦1.8, for example−0.8≦HPI_(20,24,28)≦1.8, such as HPI_(20,24,28)=−0.8.

Thus, the residues at each of positions X11, X16, X20, X24 and X28 mayindependently be Ala, Ser, Gly or Thr.

For example, each of X11 and X16 may independently be Ala or Ser, andX20, X24 and X28 may be Ala, Ser, Gly or Thr.

For example X11 and X16 may be Ala and X20, X24 and X28 may be Ala, Ser,Gly or Thr

Preferred combinations at positions X11, X16, X20, X24 and X28 mayinclude Ala/Ala/Ala/Ser/Ser, Ala/Ala/Ser/Ser/Ser, Ala/Ala/Thr/Ser/Serand Ala/Ala/Gly/Ser/Ser.

For a stomach-selective compound, within the overall criteria thatHPP≧−10, −4 or 0, it may be desirable that HPI for positions 11 and 16should independently be −3.9≦HPI_(11,16)≦3.8. For example HPI_(11,16)may be 2.8≦HPI_(11,16)≦3.8, or HPI_(11,16) may be −3.9.

For positions 20, 24 and 28 it may be desirable that HPI shouldindependently be −3.9≦HPI_(20,24,28)≦1.8. For example HPI_(26,24,28) maybe −0.8≦HPI_(20,24,28)≦1.8, or HPI_(20,24,28) may be −3.9

Thus, the residue at position X11 may be Leu, Phe or Lys.

The residue at position X16 may be Leu, Ser, Phe, Lys or Thr.

The residue at position X20 may be Ala, Ser, Leu, Gly or Thr.

The residue at position X24 may be Ala, Ser, Lys, Glu, Gly or Thr.

The residue at position X28 may be Ala, Ser, Lys, Gln, Gly or Thr.

For example, X11 and X16 may independently be Leu or Phe, X24 and X28may independently be Ala or Ser.

Preferred compounds of the present invention have at least one GLP-2biological activity, in particular in causing growth of the intestine orstomach. This can be assessed in in vivo assays, for example asdescribed in the examples, in which the mass of the intestine, or aportion thereof is determined after a test animal has been treated orexposed to a GLP-2 analogue.

The GLP-2 analogues of the present invention have one or more amino acidsubstitutions, deletions, inversions, or additions compared with nativeGLP-2 and as defined above. This definition also includes the synonymterms GLP-2 mimetics and/or GLP-2 agonists. Further, the analogue of thepresent invention may additionally have chemical modification of one ormore of its amino acid side groups, α-carbon atoms, terminal aminogroup, or terminal carboxylic acid group. A chemical modificationincludes, but is not limited to, adding chemical moieties, creating newbonds, and removing chemical moieties. Modifications at amino acid sidegroups include, without limitation, acylation of lysine ε-amino groups,N-alkylation of arginine, histidine, or lysine, alkylation of glutamicor aspartic carboxylic acid groups, and deamidation of glutamine orasparagine. Modifications of the terminal amino include, withoutlimitation, the des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include,without limitation, the amide, lower alkyl amide, dialkyl amide, andlower alkyl ester modifications. Preferably herein lower alkyl is C₁-C₄alkyl. Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled peptidechemist. The α-carbon of an amino acid may be mono- or di-methylated.

Where they are present, oxidatively stable Met-replacement amino acidmeans one which is selected among the group consisting of Met(O)(methionine sulfoxide), Met(O)₂ (methionine sulfone), Val, Ile, Asn, Glx(Glu or Gln), Tyr, Phe, Trp and preferably Leu, Nle, Ala, Ser, and Gly.

It should be understood that the peptides of the invention might also beprovided in the form of a salt or other derivative. Salts includepharmaceutically acceptable salts such as acid addition salts and basicsalts. Examples of acid addition salts include hydrochloride salts,citrate salts and acetate salts. Examples of basic salts include saltswhere the cation is selected from alkali metals, such as sodium andpotassium, alkaline earth metals, such as calcium, and ammonium ions⁺N(R³)₃(R⁴), where R³ and R⁴ independently designates optionallysubstituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl, optionallysubstituted aryl, or optionally substituted heteroaryl. Other examplesof pharmaceutically acceptable salts are described in “Remington'sPharmaceutical Sciences”, 17^(th) edition. Ed. Alfonso R. Gennaro (Ed.),Mark Publishing Company, Easton, Pa., U.S.A., 1985 and more recenteditions, and in the Encyclopaedia of Pharmaceutical Technology.

Other derivatives of the GLP-2 analogues of the invention includecoordination complexes with metal ions such as Mn²⁺ and Zn²⁺, esterssuch as in vivo hydrolysable esters, free acids or bases, hydrates,prodrugs or lipids. Esters can be formed between hydroxyl or carboxylicacid groups present in the compound and an appropriate carboxylic acidor alcohol reaction partner, using techniques well known in the art.Derivatives which as prodrugs of the compounds are convertible in vivoor in vitro into one of the parent compounds. Typically, at least one ofthe biological activities of compound will be reduced in the prodrugform of the compound, and can be activated by conversion of the prodrugto release the compound or a metabolite of it. Examples of prodrugsinclude the use of protecting groups which may be removed in situreleasing active compound or serve to inhibit clearance of the drug invivo.

When present, Z¹ and Z² each independently represent a peptide sequenceof 3-20 or 4-20 amino acid residues, e.g. in the range of 4-15, morepreferably in the range of 4-10 in particular in the range of 4-7 aminoacid residues, e.g., of 4, 5, 6 or 7 amino acid residues, such as 6amino acid residues. Each of the amino acid residues in the peptidesequences Z may independently be selected from Ala, Leu, Ser, Thr, Tyr,Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Orn. Preferably, the amino acidresidues are selected from Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg,His, Orn, and Met, as well as amino acids falling within formula I asdefined in WO01/04156, e.g., Dbu (2,4 diaminobutyric acid) or Dpr(2,3-diaminopropanoic acid), more preferably from Glu, Lys, and Met,especially Lys. The above-mentioned amino acids may have either D- orL-configuration, but preferably the above-mentioned amino acids have anL-configuration. Particularly preferred sequences Z are sequences offour, five or six consecutive lysine residues, and particularly sixconsecutive lysine residues. Exemplary sequences Z are shown in WO01/04156.

In certain embodiments, Z¹ is absent. In such cases, Z² may be eitherpresent or absent.

GLP-2 analogues having at least an EC50 of 1 nM are defined as GLP-2agonists.

The present invention includes the following peptides further describedin the experimental section below.

Particularly preferred compounds of the present invention includecompounds ZP2264, ZP2267, ZP2268 and ZP2270.

Stability Studies

The skilled person will be able to design appropriate methods (e.g.quantitative methods) for detection of degradation products of GLP-2analogues, e.g. based on those described below. Degradation may occur asoxidation, hydrolysis and deamidation, depending on the identity andposition of the amino acids in any given GLP-2 analogue, and conditionsas pH, solution and temperature. The compounds can be ranked accordingto chemical stability, when the compounds are incubated under stressedconditions (i.e. conditions likely to cause degradation) andsubsequently analysed for content of remaining intact peptide. Inaddition, the knowledge gained about major degradation products obtainedunder stressed conditions will be important for any later analyticalmethod development.

Quantitative Assays to Detect GLP Analogues

The skilled person will also be capable of designing methods (e.g.quantitative methods) for detection of GLP analogues in complexenvironments or solutions (e.g. plasma, urine, tissue homogenates, cellhomogenates, saliva or similar) to investigate the absorption,distribution, metabolism and excretion of the GLP analogues afteradministration to mammals or as part of functional studies of in vitrocell systems.

In one embodiment, a quantitative assay can be based on antibodiesraised against the GLP analogues or fragments thereof. The antibodiesobtained from the immunized animals can be used for quantitative assays.In one example a direct sandwich ELISA can be prepared using a firstantibody with affinity of one part of the molecule immobilized in amulti-well plate. The sample is then applied to the wells and the GLPanalogue is captured by the first antibody. The captured GLP analogue isthen recognized by a second antibody with affinity for another part ofthe GLP analogue. The second antibody can be labeled with an enzyme(horseradish peroxidase, alkaline phosphatase or beta-galactosidase) ora radioisotope. The amount of captured GLP analogue can then be detectedby addition of a colorimetric substrate or direct counting ofradio-emission or by scintillation. Alternatively, the amount ofcaptured GLP analogue can be detected indirectly by addition of alabeled antibody with affinity for the second antibody. Theconcentration in the sample can be estimated from the response obtainedfrom an external standard curve containing known amounts of GLPanalogue. Alternatively, the antibodies can be used to prepare a directcompetitive immuno assay, where an antibody specific for the GLPanalogue is immobilized on a multi-well plate and the sample incubatedin the wells with a predefined fixed concentration of labeled GLPanalogue. The label can be an enzyme, a fluorophore, a radioisotope orbiotin and detected using, for example, substrates (e.g. colorimetric,fluorometric or chemiluminiscent) specific for the enzymes,scintillation or avidin linked to an enzyme followed by detection asdescribed above. The amount of bound labeled GLP analogue can bedetected by an appropriate method and the concentration of GLP analoguepresent in the sample derived from the response obtained from anexternal standard curve as described above.

In another embodiment, a quantitative assay can be based on liquidchromatography tandem mass spectroscopy methodology. In such a set up,the response from a fragment specific for the GLP analogue to be studiedis monitored upon fragmentation of the parent compound induced bycollision with an inert gas (He or Ar). Prior to fragmentation thesample components can be separated by reversed phase chromatography orthe sample can be injected directly in the mass spectrometer. Ifsuitable the sample can be subjected to pretreatment (i.e., addition ofprotease inhibitors, protein precipitation, solid phase extraction,immuno-affinity extraction, etc. The concentration of GLP analoguepresent in the sample derived from the response obtained from anexternal standard curve as described above, potentially after correctionof the response using an internal standard similar to the GLP analogueto be studied.

Generation of Specific Antibodies

Specific antibodies against the GLP analogues or fragments thereof canbe induced in mammals and purified from the serum. The GLP analogues orfragments can either be used directly with an adjuvant to immunizerabbits, mice or other mammals, or the GLP analogues or fragmentsthereof can be chemically linked to a carrier molecule (i.e., keyholelimpet hemocyanin, ovalbumin, albumin etc.) and injected with anadjuvant. The injections can be repeated with 2-4 weeks intervals forextended periods to improve the affinity and selectivity of theantibodies. Polyclonal antibodies can be harvested directly from theserum. To obtain monoclonal antibodies, B cells isolated from immunizedanimals, preferably mice, should be fused with tumor cells to formantibody producing hybridomas. Screening and selection of theappropriate clones and antibodies can be performed using eitherimmobilized GLP analogues or peptides thereof followed by detection withlabeled anti-antibodies. Alternatively the screening and selection couldbe based on immobilized antibodies followed by detection with labeledGLP analogues or fragments thereof. In all cases, the label could be aradioisotope, an enzyme, a fluorophore or biotin and detected using, forexample, substrates (e.g. colorimetric, fluorometric orchemiluminiscent) specific for the enzymes, scintillation or avidinlinked to an enzyme followed by detection as described.

Synthesis of GLP-2 Analogues

It is preferred to synthesize the analogues of the invention by means ofsolid phase or liquid phase peptide synthesis. In this context,reference is given to WO 98/11125 and, amongst many others, Fields, G Bet al., 2002, “Principles and practice of solid-phase peptidesynthesis”. In: Synthetic Peptides (2^(nd) Edition) and the Examplesherein.

Thus the GLP-2 analogues may be synthesized in a number of waysincluding for example, a method which comprises:

(a) synthesizing the peptide by means of solid phase or liquid phasepeptide synthesis and recovering the synthetic peptide thus obtained; or

(b) when the peptide is constituted by naturally occurring amino acids,expressing a nucleic acid construct that encodes the peptide in a hostcell and recovering the expression product from the host cell culture;or

(c) when the peptide is constituted by naturally occurring amino acids,effecting cell-free in vitro expression of a nucleic acid construct thatencodes the peptide and recovering the expression product; or

a combination of methods of (a), (b), and (c) to obtain fragments of thepeptide, subsequently ligating the fragments to obtain the peptide, andrecovering the peptide.

Thus, for some analogues of the invention it may be advantageous toexploit genetic engineering techniques. This may be the case when thepeptide is sufficiently large (or produced as a fusion construct) andwhen the peptide only includes naturally occurring amino acids that canbe translated from RNA in living organisms.

For the purpose of recombinant gene technology nucleic acid fragmentsencoding the peptides of the invention are important chemical products.Hence, a further aspect of the present invention provides a nucleic acidmolecule comprising a nucleic acid sequence encoding a GLP-2 analogue ofthe invention, where the peptide preferably is comprised by naturallyoccurring amino acids. The nucleic acid fragments of the invention areeither DNA or RNA fragments.

The nucleic acid fragments of the invention will normally be inserted insuitable vectors to form cloning or expression vectors carrying thenucleic acid fragments of the invention; such novel vectors are alsopart of the invention. Details concerning the construction of thesevectors of the invention will be discussed in context of transformedcells and microorganisms below. The vectors can, depending on purposeand type of application, be in the form of plasmids, phages, cosmids,mini-chromosomes, or virus, but also naked DNA which is only expressedtransiently in certain cells is an important vector. Preferred cloningand expression vectors (plasmid vectors) of the invention are capable ofautonomous replication, thereby enabling high copy-numbers for thepurposes of high-level expression or high-level replication forsubsequent cloning.

The general outline of a vector of the invention comprises the followingfeatures in the 5′→3′ direction and in operable linkage: a promoter fordriving expression of the nucleic acid fragment of the invention,optionally a nucleic acid sequence encoding a leader peptide enablingsecretion (to the extracellular phase or, where applicable, into theperiplasma) of or a leader peptide for multiple use e.g. combinedsecretion, purification tag and enzymatic trimming to correct peptide orintegration into the membrane of the polypeptide fragment, the nucleicacid fragment encoding the peptide of the invention, and optionally anucleic acid sequence encoding a terminator. When operating withexpression vectors in producer strains or cell-lines it is for thepurposes of genetic stability of the transformed cell preferred that thevector when introduced into a host cell is integrated in the host cellgenome.

The vectors of the invention are used to transform host cells to producethe modified peptide of the invention. Such transformed cells, which arealso part of the invention, can be cultured cells or cell lines used forpropagation of the nucleic acid fragments and vectors of the invention,or used for recombinant production of the peptides of the invention.

Preferred transformed cells of the invention are micro-organisms such asbacteria (such as the species Escherichia (e.g. E. coli), Bacillus (e.g.Bacillus subtilis), Salmonella, or Mycobacterium (preferablynon-pathogenic, e.g. M. bovis BCG), yeasts (such as Saccharomycescerevisiae), and protozoans. Alternatively, the transformed cells arederived from a multicellular organism, i.e. it may be fungal cell, aninsect cell, a plant cell, or a mammalian cell. Also cells derived froma human being are interesting, cf. the discussion of cell lines andvectors below. For the purposes of cloning and/or optimised expressionit is preferred that the transformed cell is capable of replicating thenucleic acid fragment of the invention. Cells expressing the nucleicfragment are preferred useful embodiments of the invention; they can beused for small-scale or large-scale preparation of the peptides of theinvention.

When producing the peptide of the invention by means of transformedcells, it is convenient, although far from essential, that theexpression product is either exported out into the culture medium orcarried on the surface of the transformed cell.

When an effective producer cell has been identified it is preferred, onthe basis thereof, to establish a stable cell line which carries thevector of the invention and which expresses the nucleic acid fragmentencoding the peptide. Preferably, this stable cell line secretes orcarries the peptide of the invention, thereby facilitating purificationthereof.

In general, plasmid vectors containing replicon and control sequences,which are derived from species compatible with the host cell, are usedin connection with the hosts. The vector ordinarily carries areplication site, as well as marking sequences, which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322 (but numerous other usefulplasmids exist) a plasmid derived from an E. coli species (see, e.g.,Bolivar et al., 1977). The pBR322 plasmid contains genes for ampicillinand tetracycline resistance and thus provides easy means for identifyingtransformed cells. The pBR plasmid, or other microbial plasmid or phagemust also contain, or be modified to contain promoters, which can beused by the prokaryotic microorganism for expression.

Those promoters most commonly used in prokaryotic recombinant DNAconstruction include the β-lactamase (penicillinase) and lactosepromoter systems (Chang et al., 1978; Itakura et al., 1977; Goeddel etal., 1979) and a tryptophan (trp) promoter system (Goeddel et al., 1979;EP 0 036 776 A). While these are the most commonly used, other microbialpromoters have been discovered and utilized, and details concerningtheir nucleotide sequences have been published, enabling a skilledworker to ligate them functionally with plasmid vectors (Siebwenlist etal., 1980).

In addition to prokaryotes, eukaryotic microbes, such as yeast culturesmay also be used, and also here the promoter should be capable ofdriving expression. Saccharomyces cerevisiae, or common baker's yeast isthe most commonly used among eukaryotic microorganisms, although anumber of other strains are commonly available. For expression inSaccharomyces, the plasmid YRp7, for example, is commonly used(Stinchcomb et al., 1979; Kingsman et al., 1979; Tschemper et al.,1980). This plasmid already contains the trpl gene which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan for example ATCC No. 44076 or PEP4-1 (Jones, 1977).The presence of the trpl lesion as a characteristic of the yeast hostcell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination.

Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions are the promoter region for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedglyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible formaltose and galactose utilization. Any plasmid vector containing ayeast-compatible promoter, origin of replication and terminationsequences is suitable.

In addition to microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7 293, Spodoptera frugiperda(SF) cells (commercially available as complete expression systems fromi.a. Protein Sciences, 1000 Research Parkway, Meriden, Conn. 06450,U.S.A. and from Invitrogen), the D. melanogaster cell line S₂ availablefrom Invitrogen, PO Box 2312, 9704 CH Groningen, The Netherlands, andMDCK cell lines.

Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions on the expressionvectors are often provided by viral material. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, and most frequentlySimian Virus 40 (SV40). The early and late promoters of SV40 virus areparticularly useful because both are obtained easily from the virus as afragment, which also contains the SV40 viral origin of replication(Fiers et al., 1978). Smaller or larger SV40 fragments may also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control sequences normally associated with thedesired gene sequence, provided such control sequences are compatiblewith the host cell systems.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral (e.g. Polyoma, Adeno, VSV, BPV) or may be provided by thehost cell chromosomal replication mechanism. If the vector is integratedinto the host cell chromosome, the latter is often sufficient.

In order to obtain satisfactory yields in a recombinant productionprocess, it may be advantageous to prepare the analogues as fusionproteins, either by fusing the peptide to a fusion partner that canserve as an affinity tag (for ease of purification) and/or by havingmultiple repeats of the peptide. These methods require presence of asuitable cleavage site for a peptidase, but the skilled person will knowhow to tailor the underlying genetic constructs.

After recombinant preparation, the peptides of the invention can bepurified by methods generally known in the art, including multi-stepchromatography (ion-exchange, size-exclusion, and affinitychromatographic techniques).

Alternatively, peptides comprised of naturally occurring amino acids canbe prepared in vitro in cell free systems. This is especially expedientin cases where the peptides could be toxic for putative host cells.Thus, the present invention also contemplates use of cell-free in vitrotranslation/expression in order to prepare the peptides of theinvention. In this context, reference is made to commercially availablein vitro translation kits, materials, and technical documentation frome.g. Ambion Inc., 2130 Woodward, Austin, Tex. 78744-1832, USA.

Finally, the available methods can of course be combined so as toprepare e.g. semi-synthetic analogues. In such a set up, peptidefragments are prepared using at least 2 separate steps or methods,followed by ligation of the fragments to obtain the final peptideproduct.

Pharmaceutical Compositions and Administration

The GLP-2 analogues of the present invention, or salts or derivativesthereof, may be formulated as pharmaceutical compositions prepared forstorage or administration, and which comprise a therapeuticallyeffective amount of a GLP-2 peptide of the present invention, or a saltor derivative thereof, in a pharmaceutically acceptable carrier.

The therapeutically effective amount of a compound of the presentinvention will depend on the route of administration, the type of mammalbeing treated, and the physical characteristics of the specific mammalunder consideration. These factors and their relationship to determiningthis amount are well known to skilled practitioners in the medical arts.This amount and the method of administration can be tailored to achieveoptimal efficacy so as to deliver the peptide to the large intestine,but will depend on such factors as weight, diet, concurrent medicationand other factors, well known those skilled in the medical arts.

It is within the invention to provide a pharmaceutical composition,wherein the GLP-2 analogue, or a salt thereof is present in an amounteffective to treat or prevent stomach and bowel-related disorders.

Pharmaceutically acceptable salts of the compounds of the inventionhaving an acidic moiety can be formed using organic and inorganic bases.Suitable salts formed with bases include metal salts, such as alkalimetal or alkaline earth metal salts, for example sodium, potassium, ormagnesium salts; ammonia salts and organic amine salts, such as thoseformed with morpholine, thiomorpholine, piperidine, pyrrolidine, amono-, di- or tri-lower alkylamine (e.g., ethyl-tert-butyl-, diethyl-,diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or a mono-,di- or trihydroxy lower alkylamine (e.g., mono-, di- ortriethanolamine). Internal salts also may be formed. Similarly, when acompound of the present invention contains a basic moiety, salts can beformed using organic and inorganic acids. For example, salts can beformed from the following acids: acetic, propionic, lactic, citric,tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic,hydrochloric, hydrobromic, phosphoric, nitric, sulfuric,methanesulfonic, napthalenesulfonic, benzenesulfonic, toluenesulfonic,and camphorsulfonic as well as other known pharmaceutically acceptableacids. Amino acid addition salts can also be formed with amino acidssuch as lysine, glycine, or phenylalanine.

As is apparent to one skilled in the medical art, a “therapeuticallyeffective amount” of the peptides or pharmaceutical compositions of thepresent invention will vary depending upon the age, weight and mammalianspecies treated, the particular compounds employed, the particular modeof administration and the desired effects and the therapeuticindication. Because these factors and their relationship to determiningthis amount are well known in the medical arts, the determination oftherapeutically effective dosage levels, the amount necessary to achievethe desired result of preventing and/or treating the intestine andstomach related diseases described herein, as well as other medicalindications disclosed herein, will be within the ambit of the skilledperson.

As used herein, “a therapeutically effective amount” is one whichreduces symptoms of a given condition or pathology, and preferably whichnormalizes physiological responses in an individual with the conditionor pathology. Reduction of symptoms or normalization of physiologicalresponses can be determined using methods routine in the art and mayvary with a given condition or pathology. In one aspect, atherapeutically effective amount of one or more GLP-2 analogues orpharmaceutical composition comprising the one or more GLP-2 analogues isan amount which restores a measurable physiological parameter tosubstantially the same value (preferably to within +30%, more preferablyto within +20%, and still more preferably, to within 10% of the value)of the parameter in an individual without the condition or pathology.

In one embodiment of the invention administration of the compounds orpharmaceutical composition of the present invention is commenced atlower dosage levels, with dosage levels being increased until thedesired effect of preventing/treating the relevant medical indication,such as intestine and stomach related diseases is achieved. This woulddefine a therapeutically effective amount. For the peptides of thepresent invention, alone or as part of a pharmaceutical composition,such doses may be between about 0.01 mg/kg and 100 mg/kg body weight,such as between about 0.01 mg/kg and 10 mg/kg body weight, for examplebetween 10-100 μg/kg body weight.

For therapeutic use, the chosen GLP-2 analogue is formulated with acarrier that is pharmaceutically acceptable and is appropriate fordelivering the peptide by the chosen route of administration. For thepurpose of the present invention, peripheral parenteral routes includeintravenous, intramuscular, subcutaneous, and intra peritoneal routes ofadministration. Certain compounds used in the present invention may alsobe amenable to administration by the oral, rectal, nasal, or lowerrespiratory routes. These are so-called non-parenteral routes. Thepresent pharmaceutical composition comprises a GLP-2 analogue of theinvention, or a salt or derivative thereof and a pharmaceuticallyacceptable carrier. Suitable pharmaceutically acceptable carriers arethose used conventionally with peptide-based drugs, such as diluents,excipients and the like. Pharmaceutically acceptable carriers fortherapeutic use are well known in the pharmaceutical art, and aredescribed, for example, in Remington's Pharmaceutical Sciences, MackPublishing Co. (A. R. Gennaro edit. 1985). For example, sterile salineand phosphate-buffered saline at slightly acidic or physiological pH maybe used. pH buffering agents may be phosphate, citrate, acetate,tris/hydroxymethyl)aminomethane (TRIS),N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS),ammonium bicarbonate, diethanolamine, histidine, which is a preferredbuffer, arginine, lysine, or acetate or mixtures thereof. Preferredbuffer ranges are pH 4-8, pH 6.5-8, more preferably pH 7-7.5.Preservatives, such as para, meta, and ortho-cresol, methyl- andpropylparaben, phenol, benzyl alcohol, sodium benzoate, benzoic acid,benzyl-benzoate, sorbic acid, propanoic acid, esters of p-hydroxybenzoicacid may be provided in the pharmaceutical composition. Stabilizers,preventing oxidation, deamidation, isomerisation, racemisation,cyclisation, peptide hydrolysis, such as e.g. ascorbic acid, methionine,tryptophane, EDTA, asparagine, lysine, arginine, glutamine and glycinemay be provided in the pharmaceutical composition. Stabilizers,preventing aggregation, fibrillation and precipitation, such as Sodiumdodecyl sulphate, polyethylene glycol, carboxymethyl cellulose,cyclodextrine may be provided in the pharmaceutical composition. Organicmodifiers for solubilization or preventing aggregation, such as ethanol,acetic acid or acetate and salts thereof may be provided in thepharmaceutical composition. Isotonicity makers such as salts e.g. sodiumchloride or most preferred carbohydrates e.g. dextrose, mannitol,lactose, trehalose, sucrose or mixtures thereof may be provided in thepharmaceutical composition.

Detergents, such as Tween 20, Tween 80, SDS, Poloxamers e.g. PluronicF-68, Pluronic F-127, may be provided in the pharmaceutical composition.Dyes and even flavoring agents may be provided in the pharmaceuticalcomposition. In another embodiment, a pharmaceutically acceptable acidaddition salt of the GLP-2 peptide analogue is provided for. Suspendingagents may be used.

Organic modifiers, such as ethanol, tertiary-buthanol, 2-propanol,ethanol, glycerol, Polyethylene glycol may be provided in thepharmaceutical formulation for lyophilization of a lyophilized product.Bulking agents and isotonicity makers such as salt e.g. sodium chloride,carbohydrates e.g. dextrose, mannitol, lactose, trehalose, sucrose ormixtures thereof, aminoacids e.g. glycine, glutamate, or excipients suchas cystein, lecithin or human serum albumin, or mixtures thereof may beprovided in the pharmaceutical composition for lyophilization.

The pharmaceutical compositions of the present invention may beformulated and used as tablets, capsules or elixirs for oraladministration; suppositories for rectal administration; preferablysterile solutions or sterile powder or suspensions for injectableadministration; and the like. The dose and method of administration canbe tailored to achieve optimal efficacy but will depend on such factorsas weight, diet, concurrent medication and other factors, which thoseskilled in the medical arts will recognize.

When administration is to be parenteral, such as intravenous andsubcutaneous, e.g., on a daily basis, injectable pharmaceuticalcompositions can be prepared in conventional forms, either as aqueoussolutions or suspensions; lyophilized, solid forms suitable forreconstitution immediately before use or suspension in liquid prior toinjection, or as emulsions.

Diluents for reconstitution of the lyophilized product may be a suitablebuffer from the list above, water, saline, dextrose, mannitol, lactose,trehalose, sucrose, lecithin, albumin, sodium glutamate, cysteinehydrochloride; or water for injection with addition of detergents, suchas Tween 20, Tween 80, poloxamers e.g. pluronic F-68 or pluronic F-127,polyethylene glycol, and or with addition of preservatives such aspara-, meta-, and ortho-cresol, methyl- and propylparaben, phenol,benzyl alcohol, sodium benzoate, benzoic acid, benzyl-benzoate, sorbicacid, propanoic acid, esters of p-hydroxybenzoic acid, and or withaddition of an organic modifier such as ethanol, acitic acid, citricacid, lactic acid or salts thereof.

In addition, if desired, the injectable pharmaceutical compositions maycontain minor amounts of non-toxic auxiliary substances, such as wettingagents, or pH buffering agents. Absorption enhancing preparations (e.g.,liposomes, detergents and organic acids) may be utilized.

In one embodiment of the invention, the compounds are formulated foradministration by infusion, e.g., when used as liquid nutritionalsupplements for patients on total parenteral nutrition therapy (forexample neonatals, or patients suffering from cachexia or anorexia), orby injection, for example subcutaneously, intraperitoneal orintravenously, and are accordingly utilized as aqueous solutions insterile and pyrogen-free form and optionally buffered to physiologicallytolerable pH, e.g., a slightly acidic or physiological pH. Formulationfor intramuscular administration may be based on solutions orsuspensions in plant oil, e.g. canola oil, corn oil or soy bean oil.These oil based formulations may be stabilized by antioxidants e.g. BHA(butylated hydroxianisole) and BHT (butylated hydroxytoluene).

Thus, the present peptide compounds may be administered in a vehicle,such as distilled water or in saline, phosphate buffered saline, 5%dextrose solutions or oils. The solubility of the GLP-2 analogue may beenhanced, if desired, by incorporating a solubility enhancer, such asdetergents and emulsifiers.

The aqueous carrier or vehicle can be supplemented for use asinjectables with an amount of gelatin that serves to depot the GLP-2analogue at or near the site of injection, for its slow release to thedesired site of action. Alternative gelling agents, such as hyaluronicacid, may also be useful as depot agents.

In one embodiment of the present invention the formulation comprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 mM to 300 mM, preferably from 3 to 200 mM, most preferably from 20to 100 mM;

b. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; and

c. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In another embodiment of the present invention the formulation comprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 mM to 300 mM, preferably from 3 to 200 mM, most preferably from 20to 100 mM L-histidine;

b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;

c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; and

d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In still another embodiment of the present invention the formulationcomprises

a. L-histidine dissolved in water to obtain final concentrations of upto 200 mM, preferably from 3 to 100 mM, most preferably from 5 to 50 mML-histidine;

b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;

c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; and

d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In yet another embodiment of the present invention the formulationcomprises

a. L-histidine dissolved in water to obtain final concentrations of from0.5 to 300 mM, preferably from 3 to 200 mM, most preferably from 20 to100 mM L-histidine;

b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;

c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; and

d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In yet another embodiment of the present invention the formulationcomprises

a. L-histidine dissolved in water to obtain final concentrations of fromup to 200 mM, preferably from 3 to 100 mM, most preferably from 5 to 50mM L-histidine;

b. L-Arginine to obtain up to 200 mM, preferably from 0.5 to 100 mM,most preferably from 5 to 50 mM;

c. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM; and

d. acetic acid to obtain up to 200 mM, preferably from 0.05 to 100 mM,most preferably from 0.5 to 50 mM into solution.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product.

In yet another embodiment of the present invention the formulationcomprises

a. N-acetate dissolved in water to obtain final concentrations of fromup to 200 mM, preferably from 0.5 to 100 mM, most preferably from 5 to50 mM L-histidine;

b. mannitol to obtain up to 350 mM, preferably from 30 to 300 mM, mostpreferably from 100 mM to 230 mM.

Appropriate amount of therapeutic compound is added to obtainconcentrations of from 1 to 100 mg/mL, preferably from 5 to 50 mg/mL,most preferably from 10 to 30 mg/mL.

pH are adjusted to final pH at from 4 to 8, preferably from 6.5 to 7.5,most preferably from 6.7 to 7.3. The resulting solution is adjusted totarget weight, sterile filtered and dispensed into appropriate aliquotsin vials for pharmaceutical use. The formulation is further processedaccording to a liquid product or to a lyophilised product

The GLP-2 analogues of the invention may also be formulated as a slowrelease implantation device for extended and sustained administration ofthe GLP-2 peptide analogue. Such sustained release formulations may bein the form of a patch positioned externally on the body. Examples ofsustained release formulations include composites of biocompatiblepolymers, such as poly(lactic acid), poly(lactic-co-glycolic acid),methylcellulose, hyaluronic acid, sialic acid, silicate, collagen,liposomes and the like. Sustained release formulations may be ofparticular interest when it is desirable to provide a high localconcentration of a GLP-2 analogue of the invention.

The GLP-2 analogue may be utilized in the form of a sterile-filled vialor ampoule containing an intestinotrophic amount of the peptide, ineither unit dose or multi-dose amounts. The vial or ampoule may containthe GLP-2 analogue and the desired carrier, as an administration readyformulation. Alternatively, the vial or ampoule may contain the GLP-2peptide in a form, such as a lyophilized form, suitable forreconstitution in a suitable carrier, such as sterile water orphosphate-buffered saline.

As an alternative to injectable formulations, the GLP-2 analogue may beformulated for administration by other routes. Oral dosage forms, suchas tablets, capsules and the like, can be formulated in accordance withstandard pharmaceutical practice. According to the present invention,the GLP-2 analogue is administered to treat individuals that wouldbenefit from growth of small bowel tissue.

Nasal dosage forms can be formulated with addition of enhancers, such asChitosan or detergents such as Tween 20, Tween 80, Poloxamers e.g.Pluronic F-68, Pluronic F-127; Brij 35, Brij 72, cremophor EL.

The peptide compounds of the present invention may be used alone, or incombination with compounds having an anti-inflammatory effect. Withoutbeing bound by theory it is envisioned that such combination treatmentmay enforce the beneficial treatment effects of the present peptideanalogues.

The therapeutic dosing and regimen most appropriate for patienttreatment will of course vary with the disease or condition to betreated, and according to the patient's weight and other parameters.Without wishing to be bound by any particular theory, it is expectedthat doses, in the μg/kg range, and shorter or longer duration orfrequency of treatment may produce therapeutically useful results, suchas a statistically significant increase particularly in small bowelmass. In some instances, the therapeutic regimen may include theadministration of maintenance doses appropriate for preventing tissueregression that occurs following cessation of initial treatment. Thedosage sizes and dosing regimen most appropriate for human use may beguided by the results obtained by the present invention, and may beconfirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined byconventional means, starting with a low dose in laboratory animals andthen increasing the dosage while monitoring the effects, andsystematically varying the dosage regimen as well. Numerous factors maybe taken into consideration by a clinician when determining an optimaldosage for a given subject. Such considerations are known to the skilledperson.

A human dose of a GLP-2 peptide according to the invention may in oneembodiment be from about 10 μg/kg body weight/day to about 10 mg/kg/day,preferably from about 50 μg/kg/day to about 5 mg/kg/day, and mostpreferably about 100 μg/kg/day to 1 mg/kg/day.

Medical Conditions

The peptides of the present invention are useful as a pharmaceuticalagent for preventing or treating an individual suffering fromgastro-intestinal disorders, including the upper gastrointestinal tractof the oesophagus by administering an effective amount of a GLP-2analogue, or a salt thereof as described herein. The stomach andintestinal-related disorders include ulcers of any aetiology (e.g.,peptic ulcers, Zollinger-Ellison Syndrome, drug-induced ulcers, ulcersrelated to infections or other pathogens), digestion disorders,malabsorption syndromes, short-bowel syndrome, cul-de-sac syndrome,inflammatory bowel disease (Crohns disease and ulcerative colitis),celiac sprue (for example arising from gluten induced enteropathy orceliac disease), tropical sprue, hypogammaglobulinemic sprue, andchemotherapy and/or radiation hemotherapy induced mucositis anddiarrhea.

For patients having gastrointestinal mucosal neoplasia, or an increasedrisk of gastrointestinal mucosal neoplasia, it may be desirable toselect a compound so as to reduce or abrogate the risk of reduced sideeffects such as stimulation or aggravation of gastrointestinal mucosalneoplasia. For example, when selecting a compound for treating a patientwith colon neoplasia (whether benign or malignant), or at risk ofdeveloping colon neoplasia, it may be more appropriate to select acompound which is selective for the small intestine over the colon thana non-selective compound or a compound which is selective for the colonover the small intestine

As mentioned above in general, individuals who would benefit fromincreased small intestinal mass and consequent and/or maintenance ofnormal small intestine mucosal structure and function are candidates fortreatment with the present GLP-2 analogues. Particular conditions thatmay be treated with GLP-2 analogue include the various forms of sprueincluding celiac sprue which results from a toxic reaction toalpha-gliadin from heat and may be a result of gluten-inducedenteropathy or celiac disease, and is marked by a significant loss ofvillae of the small bowel; tropical sprue which results from infectionand is marked by partial flattening of the vitae; hypogammaglobulinemicsprue which is observed commonly in patients with common variableimmunodeficiency or hypogammaglobulinemia and is marked by significantdecrease in villus height. The therapeutic efficacy of the GLP-2analogue treatment may be monitored by enteric biopsy to examine thevillus morphology, by biochemical assessment of nutrient, absorption, bynon invasive determination of intestinal permeability, by patient weightgain, or by amelioration of the symptoms associated with theseconditions.

The GLP-2 analogues of the present invention may be useful aspharmaceutical agents for preventing or treating stomach relateddisorders including ulcers of any aetiology (e.g., peptic ulcers,Zollinger-Ellison Syndrome, drug-induced ulcers, ulcers related toinfections or other pathogens).

Other conditions that may be treated with the GLP-2 analogues of theinvention, or for which the GLP-2 analogues may be usefulprophylactically, include in addition to the above mentioned radiationenteritis, infectious or post-infectious enteritis, and small intestinaldamage due to cancer-chemotherapeutic or toxic agents.

The GLP-2 analogues may also be used for the treatment of malnutrition,for example cachexia and anorexia.

A particular embodiment of the invention is concerned with using thepresent peptides for the prevention and/or treatment of intestinaldamage and dysfunction. The stem cells of the small intestinal mucosaare particularly susceptible to the cytotoxic effects of chemotherapydue to their rapid rate of proliferation (Keefe et al., Gut 2000; 47:632-7). Administration of the present GLP-2 peptide agonists may enhancetrophic effect in the intestinal crypts and rapidly provide new cells toreplace the damaged intestinal epithelium following chemotherapy and/orradiation therapy. The ultimate goal achieved by administering thepresent peptides is to reduce the morbidity related to gastrointestinaldamage of patients undergoing chemotherapy treatment while increasingtolerance to more aggressive chemotherapy, radiation and combinationchemotherapy and radiation therapies. Concomitant prophylactic ortherapeutic treatment may be provided in accordance with the presentinvention to patients undergoing or about to undergo radiation therapy.

Gastrointestinal mucositis after anti-cancer chemotherapy is anincreasing problem that is essentially untreatable once established,although it gradually remits. Studies conducted with the commonly usedcytostatic cancer drugs 5-FU and irinotecan have demonstrated thateffective chemotherapy with these drugs predominantly affects structuralintegrity and function of the small intestine while the colon is lesssensitive and mainly responds with increased mucus formation (Gibson etal., J Gastroenterol Hepatol. 18(9):1095-1100, 2003; Tamaki et al., JInt Med Res. 31(1):6-16, 2003).

The novel GLP-2 analogues of the present invention may be useful in theprevention and/or treatment of gastrointestinal injury and side effectsof chemotherapeutic agents. This potentially important therapeuticapplication may apply to currently used chemotherapeutic agents such asbut not limited to: 5-FU, Altretamine, Bleomycin, Busulfan,Capecitabine, Carboplatin, Carmustine, Chlorambucil, Cisplatin,Cladribine, Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine,Dactinomycin, Daunorubicin, Docetaxel, Doxorubicin, Epirubicin,Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxycarbamide,Idarubicin, Ifosfamide, Irinotecan, Liposomal doxorubicin, Leucovorin,Lomustine, Melphalan, Mercaptopurine, Mesna, Methotrexate, Mitomycin,Mitoxantrone, Oxaliplatin, Paclitaxel, Pemetrexed, Pentostatin,Procarbazine, Raltitrexed, Streptozocin, Tegafur-uracil, Temozolomide,Thiotepa, Tioguanine/Thioguanine, Topotecan, Treosulfan, Vinblastine,Vincristine, Vindesine, Vinorelbine, Bleomycin, Busulfan, Capecitabine,Carboplatin, Carmustine, Chlorambucil, Cisplatin, Cladribine,Crisantaspase, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin,Daunorubicin, Docetaxel, Doxorubicin, Epirubicin, Etoposide,Fludarabine, Gemcitabine, Hydroxycarbamide, Idarubicin, Ifosfamide,Irinotecan, Liposomal doxorubicin, Leucovorin, Lomustine, Melphalan,Mercaptopurine, Methotrexate, Mitomycin, Mitoxantrone, Oxaliplatin,Paclitaxel, Pemetrexed, Pentostatin, Procarbazine, Raltitrexed,Streptozocin, Tegafur-uracil, Temozolomide, Thiotepa,Tioguanine/Thioguanine, Topotecan, Treosulfan, Vinblastine, Vincristine,Vindesine, and Vinorelbine.

It is envisioned that the present peptides may be employed in a methodof treating neo-natals by administering an effective amount of a GLP-2analogue, or a salt thereof. Complications with feeding neonatals due tothe lack of development of the intestine may be overcome by using thepresent peptide agonists.

In another embodiment the invention describes a method of treatingDPP-IV (dipeptidylpeptidase-IV) mediated conditions by administering toa patient in need thereof an effective amount of a GLP-2 analogue, or asalt thereof. Such diseases include conditions in which the DPP-IVenzyme is over expressed.

The pharmaceutical composition may in one embodiment be formulated tocause slow release of said GLP-2 analogue, or a salt or derivativethereof as described above.

It is envisaged that the present peptides may be employed in a method oftreating neo-natals by administering an effective amount of a GLP-2analogue, or a salt thereof. Complications with feeding neonatals due tothe lack of development of the intestine may be overcome by using thepresent peptide agonists.

In another embodiment the invention describes a method of treatingDPP-IV (dipeptidylpeptidase-IV) mediated conditions by administering toa patient in need thereof an effective amount of a GLP-2 analogue, or asalt thereof. Such diseases include conditions in which the DPP-IVenzyme is over expressed.

EXAMPLES

The following examples are provided to illustrate preferred aspects ofthe invention and are not intended to limit the scope of the invention.

General Peptide Synthesis

Apparatus and Synthetic Strategy

Peptides were synthesized batchwise in a polyethylene vessel equippedwith a polypropylene filter for filtration using9-fluorenylmethyloxycarbonyl (Fmoc) as N-α-amino protecting group andsuitable common protection groups for side-chain functionalities.

Solvents

Solvent DMF (N,N-dimethylformamide, Riedel de-Häen, Germany) waspurified by passing through a column packed with a strong cationexchange resin (Lewatit S 100 MB/H strong acid, Bayer AG Leverkusen,Germany) and analyzed for free amines prior to use by addition of3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH) giving rise toa yellow color (Dhbt-O-anion) if free amines are present. Solvent DCM(dichloromethane, analytical grade, Riedel de-Häen, Germany) was useddirectly without purification. Acetonitril (HPLC-grade, Lab-Scan, DublinIreland) was used directly without purification.

Amino Acids

Fmoc-protected amino acids were purchased from Advanced ChemTech (ACT)in suitable side-chain protected forms.

Coupling Reagents

Coupling reagent diisopropylcarbodiimide (DIC) was purchased from Riedelde-Häen, Germany.

Solid Supports

Peptides were synthesized on TentaGel S resins 0.22-0.31 mmol/g.TentaGel S-Ram, TentaGel S RAM-Lys(Boc)Fmoc (Rapp polymere, Germany)were used in cases where a C-terminal amidated peptide was preferred,while TentaGel S PHB, TentaGel S PHB Lys(Boc)Fmoc were used when aC-terminal free carboxylic acid was preferred.

Catalysts and Other Reagents

Diisopropylethylamine (DIEA) was purchased from Aldrich, Germany,piperidine and pyridine from Riedel-de Häen, Frankfurt, Germany.Ethandithiol was purchased from Riedel-de Häen, Frankfurt, Germany.3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (Dhbt-OH),1-hydroxybenzotriazole (HOBt) (HOAt) were obtained from Fluka,Switzerland. Acetic anhydride was obtained from Fluka.

Coupling Procedures

The amino acids were coupled as in situ generated HObt or HOAt estersmade from appropriate N-a-protected amino acids and HObt or HOAt bymeans of DIC in DMF. Acylations were checked by the ninhydrin testperformed at 80 oC in order to prevent Fmoc deprotection during the test(Larsen, B. D. and Holm, A., Int. J. Peptide Protein Res. 43, 1994,1-9).

Deprotection of the N-α-Amino Protecting Group (Fmoc).

Deprotection of the Fmoc group was performed by treatment with 20%piperidine in DMF (1×5 and 1×10 min.), followed by wash with DMF (5×15ml, 5 min. each) until no yellow color could be detected after additionof Dhbt-OH to the drained DMF.

Coupling of HOBt-Esters

3 eq. N-α-amino protected amino acid was dissolved in DMF together with3 eq. HObt and 3 eq DIC and then added to the resin.

Cleavage of Peptide from Resin with Acid.

Peptides were cleaved from the resins by treatment with 95%triflouroacetic acid (TFA, Riedel-de Häen, Frankfurt, Germany)-water v/vor with 95% TFA and 5% ethandithiol v/v at r.t. for 2 h. The filteredresins were washed with 95% TFA-water and filtrates and washingsevaporated under reduced pressure. The residue was washed with ether andfreeze dried from acetic acid-water. The crude freeze dried product wasanalyzed by high-performance liquid chromatography (HPLC) and identifiedby mass spectrometry (MS).

Batchwise Peptide Synthesis on TentaGel Resin (PEG-PS).

TentaGel resin (1 g, 0.23-0.24 mmol/g) was placed in a polyethylenevessel equipped with a polypropylene filter for filtration. The resinwas swelled in DMF (15 ml), and treated with 20% piperidine in DMF inorder to remove the initial Fmoc group either on the linker TentaGel SRAM or on the first amino acid on the resin TentaGel S RAM-Lys(Boc)Fmoc.The resin was drained and washed with DMF until no yellow color could bedetected after addition of Dhbt-OH to the drained DMF. The amino acidsaccording to the sequence were coupled as preformed Fmoc-protected HObtesters (3 eq.) as described above. The couplings were continued for 2 h,unless otherwise specified. The resin was drained and washed with DMF(5×15 ml, 5 min each) in order to remove excess reagent. All acylationswere checked by the ninhydrin test as described above. After completedsynthesis the peptide-resin was washed with DMF (3×15 ml, 5 min each),DCM (3×15 ml, 1 min each) and finally diethyl ether (3×15 ml, 1 mineach) and dried in vacuo. The peptide was cleaved from the resin asdescribed earlier and the crude peptide product was analysed andpurified as described below

HPLC Conditions

Gradient HPLC analysis was done using a Hewlett Packard HP 1100 HPLCsystem consisting of a HP 1100 Quaternary Pump, a HP 1100 Autosampler aHP 1100 Column Thermostat and HP 1100 Multiple Wavelength Detector.Hewlett Packard Chemstation for LC software (rev. A.06.01) was used forinstrument control and data acquisition. The following columns and HPLCbuffer system was used:

Column: VYDAC 238TP5415, C-18, 5 mm, 300 Å 150×4.6 mm.

Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90% MeCN.

Gradient: 0-1.5 min. 0% B

-   -   1.5-25 min 50% B    -   25-30 min 100% B    -   30-35 min 100% B    -   35-40 min 0% B

Flow 1, ml/min, oven temperature 40 oC, UV detection: I=215 nm.

HPLC Purification of the Crude Peptide

The crude peptide products were purified PerSeptive Biosystems VISIONWorkstation. VISION 3.0 software was used for instrument control anddata acquisition. The following column and HPLC buffer system was used:

Column: Kromasil KR 100 Å, 10 mm C-8, 250×50.8 mm.

Buffer system: Buffers: A: 0.1% TFA in MQV; B: 0.085% TFA, 10% MQV, 90%MeCN.

Gradient: 0-37 min. 0-40% B

Flow 35 ml/min, UV detection: I=215 nm and 280 nm.

Mass Spectroscopy

The peptides were dissolved in super gradient methanol (Labscan, Dublin,Ireland), milli-Q water (Millipore, Bedford, Mass.) and formic acid(Merck, Damstadt, Germany) (50:50:0.1 v/v/v) to give concentrationsbetween 1 and 10 mg/ml. The peptide solutions (20 ml) were analysed inpositive polarity mode by ESI-TOF-MS using a LCT mass spectrometer(Micromass, Manchester, UK) accuracy of +/−0.1 m/z.

General Synthetic Procedure

In all syntheses dry TentaGel-S-Ram resin (1 g, 0.22-0.31 mmol/g) wasplaced in a polyethylene vessel equipped with a polypropylene filter forfiltration. The resin was swelled in DMF (15 ml), and treated with 20%piperidine in DMF to secure the presence of non-protonated amino groupson the resin. The resin was drained and washed with DMF until no yellowcolour could be detected after addition of Dhbt-OH to the drained DMF.The amino acids according to the sequence were coupled as preformedFmoc-protected HOBt esters (3 eq.) as described above. The couplingswere continued for 2 h, unless otherwise specified. The resin wasdrained and washed with DMF (5×15 ml, 5 min each) in order to removeexcess reagent. All acylations were checked by the ninhydrin testperformed at 80° C. After completed synthesis the peptide-resin waswashed with DMF (3×15 ml, 5 min each), DCM (3×15 ml, 1 min each) andfinally diethyl ether (3×15 ml, 1 min each) and dried in vacuo. Thepeptide was then cleaved from the resin as described above and freezedried.

After purification using preparative HPLC as described above, thepeptide product was collected and the identity of the peptide wasconfirmed by ES-MS. This procedure was used for the synthesis of allpeptides exemplified further below.

Compounds Synthesised

Using the above techniques compounds 1809 to 1861 and reference compound1559 (H-[Gly2]hGLP-2-OH) were synthesised using the methods describedabove (Table 1).

TABLE 1 Compounds synthesized Mw mono Mw mono isotopic isotopic Yield/gZP (g/mol) (g/mol) Purity resin no. Sequence calculated found % mg 2264H-HGEGSFSDELATILEALAAADFIAWLIATKITD-NH2 3487.79 3487.5 93 21 2266H-HGEGSFSDELETILEELAAEDFIEWLIETKITD-NH2 3777.81 3777.1 88 38 2267H-HGEGSFSDELSTILESLAAADFIAWLIATKITD-NH2 3519.78 3519.5 96 30 2268H-HGEGSFSDELATILEALAASDFISWLISTKITD-NH2 3535.77 3535.68 91 17 2269H-HGEGSFSDELKTILESLAAADFIEWLIQTKITD-NH2 3675.87 3675.4 94 34 2272H-HGEGSFSDELATILESLAAADFISWLIATKITD-NH2 3519.78 3519.6 91 20 2263H-HGEGSFSDELSTILESLAASDFISWLISTKITD-NH2 3567.76 3567.3 62 16 2270H-HGEGSFSDELNTILESLAASDFISWLISTKITD-NH2 3594.77 3594.5 74 28 2242H-HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH 3675.19 3675.9 88 87 2378H-HGEGSFSDELETILEELAAEDFIEWLIETKITDKKKKKK-NH2 4546.38 4546.50 93 73.42379 H-HGEGSFSDELKTILESLAAADFIEWLIQTKITDKKKKKK-NH2 4444.44 4444.13 9624.9 2380 H-HGEGSFSDELSTILESLAASDFISWLISTKITDKKKKKK-NH2 4336.33 4336.7596 40.6 2381 H-HGEGSFSDELATILEALAASDFISWLISTKITDKKKKKK-NH2 4304.344304.50 97 38.5 2382 H-HGEGSFSDELNTILESLAASDFISWLISTKITDKKKKKK-NH24363.34 4363.55 94 12.9 2383H-HGEGSFSDELNTILESLAARDFISWLISTKITDKKKKKK-NH2 4432.41 4432.60 96 16.82384 H-HGEGSFSDELATILEALAAADFIAWLISTKITDKKKKKK-NH2 4272.35 4272.40 9827.3 2385 H-HGEGSFSDELSTILEALAASDFISWLISTK1TDKKKKKK-NH2 4320.34 4320.5095 40.5 2386 H-HGEGSFSDELATILESLAASDFISWLISTKITDKKKKKK-NH2 4320.344320.75 89 65.3 2394 H-HGEGSFSDELLTILELLAASDFISWLISTKITDKKKKKK-NH24388.44 4389.13 90 23.2 2395H-HGEGSFSDELKTILEKLAAKDFIKWLIKTKITDKKKKKK-NH2 4541.65 4541.61 96 32 2396H-HGEGSFSDELFTILEFLAASDFISWLISTKITDKKKKKK-NH2 4456.75 4456.40 93 21.52397 H-HGEGSFSDELATILEALAAADFIAWLISTKITD-NH2 3503.88 3503.78 92 73.52398 H-HGEGSFSDELSTILEALAASDFISWLISTKITD-NH2 3551.77 3552.00 89 23.52399 H-HGEGSFSDELATILESLAASDFISWLISTKITD-NH2 3551.77 3551.75 80 45.62400 H-HGEGSFSDELLTILELLAASDFISWLISTKITD-NH2 3619.87 3620.50 84 6.8 2401H-HGEGSFSDELFTILEFLAASDFISWLISTKITD-NH2 3687.83 3688.00 75 11.9 2402H-HGEGSFSDELATILEALAAADFIAWLIATKITDKKKKKK-NH2 4256.36 4256.88 97 29.92403 H-HGEGSFSDELSTILESLAAADFIAWLIATKITDKKKKKK-NH2 4288.35 4288.63 9348.4 2404 H-HGEGSFSDELSTILEALAAADFIAWLIATKITDKKKKKK-NH2 4272.35 4272.6392 28.5 2411 H-HGEGSFSDELATILEALAAADFISWLIATKITDKKKKKK-NH2 4272.354272.88 93 35.5 2412 H-HGEGSFSDELLTILELLAAADFIAWLIATKITDKKKKKK-NH24340.45 4341.13 88 58.7 2413H-HGEGSFSDELATILESLAAADFISWLIATKITDKKKKKK-NH2 4288.35 4288.88 95 52.32414 H-HGEGSFSDELATILESLAAADFIAWLIATKITD-NH2 3503.78 3504.25 91 49.52415 H-HGEGSFSDELATILESLAAADFIAWLIATKITDKKKKKK-NH2 4272.35 4272.88 9843.25 2416 H-HGEGSFSDELLTILELLAALDFIAWLIATKITDKKKKKK-NH2 4382.50 4383.0088 26.5 2417 H-HGEGSFSDELATILEALAASDFIAWLIATKITD-NH2 3503.78 3504.10 9858.8 2418 H-HGEGSFSDELATILEALAAADFISWLIATKITD-NH2 3503.78 3504.00 9783.3 2420 H-HGEGSFSDELSTILEALAAADFIAWLIATKITD-NH2 3503.78 3504.63 9462.2 2423 H-HGEGSFSDELATILEALAASDFIAWLIATKITDKKKKKK-NH2 4272.35 4273.5093 27.2 2424 H-HGEGSFSDELLTILELLAALDFILWLILTKITDKKKKKK-NH2 4466.59 4467.00 78 29

Example 1 Synthesis of Compound 2264

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ala-Leu-Ala-Ala-Ala-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-NH₂on TentaGel S RAM.

Dry TentaGel S RAM (0.2 mmol/g, 1 g) was placed in a polyethylene vesselequipped with a polypropylene filter for filtration and treated asdescribed under “batchwise peptide synthesis on TentaGel resin” untilfinishing the coupling of the N-terminal Histidine. All couplings werecontinued over night. The acylations were checked as earlier described.After completed synthesis and deprotection of the N-terminal Fmoc groupthe peptide was cleaved from the resin as described above. Afterpurification using preparative HPLC as earlier described, 413 mg peptideproduct was collected with a purity better than 93% and the identity ofthe peptide was confirmed by MS (found M 3488,13, calculated M 3487,79).

Example 2 Synthesis of Compound 2268

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ala-Leu-Ala-Ala-Ser-Asp-Phe-Ile-Ser-Trp-Leu-Ile-Ser-Thr-Lys-Ile-Thr-Asp-NH₂on TentaGel S RAM.

Dry TentaGel S RAM (0.2 mmol/g, 1 g) was placed in a polyethylene vesselequipped with a polypropylene filter for filtration and treated asdescribed under “batchwise peptide synthesis on TentaGel resin” untilfinishing the coupling of the N-terminal Histidine. All couplings werecontinued over night. The acylations were checked as earlier described.After completed synthesis and deprotection of the N-terminal Fmoc groupthe peptide was cleaved from the resin as described above. Afterpurification using preparative HPLC as earlier described, 132 mg peptideproduct was collected with a purity better than 91% and the identity ofthe peptide was confirmed by MS (found M 3535,88, calculated M 3535,77).

Example 3 Synthesis of Compound 2264(Lys6)

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ala-Leu-Ala-Ala-Ala-Asp-Phe-Ile-Ala-Trp-Leu-Ile-Ala-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-LysNH₂ on TentaGel S RAM-Lys(Boc)-Fmoc.

Dry TentaGel S RAM-Lys(Boc)-Fmoc (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, the peptide product was collected with a purity better than90% and the identity of the peptide was confirmed by MS (found M 4256,40calculated M 4256,36).

Example 4 Synthesis of Compound 2268(Lys6)

H-His-Gly-Glu-Gly-Ser-Phe-Ser-Asp-Glu-Leu-Ala-Thr-Ile-Leu-Glu-Ala-Leu-Ala-Ala-Ser-Asp-Phe-Ile-Ser-Trp-Leu-Ile-Ser-Thr-Lys-Ile-Thr-Asp-Lys-Lys-Lys-Lys-Lys-LysNH₂ on TentaGel S RAM-Lys(Boc)-Fmoc.

Dry TentaGel S RAM-Lys(Boc)-Fmoc (0.24 mmol/g, 1 g) was placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand treated as described under “batchwise peptide synthesis on TentaGelresin” until finishing the coupling of the N-terminal Histidine. Allcouplings were continued over night. The acylations were checked asearlier described. After completed synthesis and deprotection of theN-terminal Fmoc group the peptide was cleaved from the resin asdescribed above. After purification using preparative HPLC as earlierdescribed, the peptide product was collected with a purity better than90% and the identity of the peptide was confirmed by MS (found M 4304,10calculated M 4304,34).

Example 5 Synthesis of Compound 2264 A (Acetate Salt)

Counter ion exchange from trifluoroacetate to acetate of Compound 2264.

The purified synthetic peptide product of compound 2264 is isolated as atrifluoroacetate salt, due to the presence of trifluoroacetic acid (0.1%v/v) in the HPLC buffers used for the purification of the crudesynthetic peptide product.

In order to exchange the counter ion trifluoroacetate with acetate, asolution of the peptide was passed through a column packed with strongbase ion exchange resin on the acetate (Dowex 1×8). Compound 2264T isdissolved in water. The solution is passed through a column containingstrong base ion exchange resin on the acetate (Dowex 1×8; capacity 1.33meq/ml resin). The resin is then washed with water and the eluate iscollected and lyophilized resulting in the acetate salt with a puritybetter than 90%.

Example 6 Synthesis of Compound 2264 C (Chloride Salt)

Counter ion exchange from trifluoroacetate (Tfa) to chloride (Cl—) ofCompound 2264.

Compound 2264T was dissolved in 0.1M hydrochloric acid and the resultingsolution was lyophilized. The remanence was dissolved in water andlyophilized again resulting in the chloride salt with a purity betterthan 90%.

Further selective GLP-2 analogue compounds are listed in table 2

TABLE 2 List of selective GLP-2 analogue compounds. Compound 1 2 3 4 5 67 8 9 10 11 12 13 14 15 16 17 18 1 H G E G S F S D E L A T I L E A L A 2H G E G S F S D E L L T I L E L L A 3 H G E G S F S D E L F T I L E F LA 4 H G E G S F S D E L V T I L E V L A 5 H G E G S F S D E L I T I L EI L A 6 H G E G S F S D E L L T I L E L L A 7 H G E G S F S D E L I T IL E I L A 8 H G E G S F S D E L V T I L E V L A 9 H G E G S F S D E L FT I L E F L A 10 H G E G S F S D E L A T I L E A L A 11 H G E G S F S DE L L T I L E L L A 12 H G E G S F S D E L I T I L E I L A 13 H G E G SF S D E L V T I L E V L A 14 H G E G S F S D E L F T I L E F L A 15 H GE G S F S D E L A T I L E A L A 16 H G E G S F S D E L A T I L E A L A17 H G E G S F S D E L A T I L E A L A 18 H G E G S F S D E L A T I L EA L A 19 H G E G S F S D E L A T I L E A L A 20 H G E G S F S D E L A TI L E A L A 21 H G E G S F S D E L A T I L E A L A 22 H G E G S F S D EL S T I L E S L A 23 H G E G S F S D E L A T I L E S L A 24 H G E G S FS D E L S T I L E A L A 25 H G E G S F S D E L A T I L E S L A 26 H G EG S F S D E L S T I L E S L A 27 H G E G S F S D E L A T I L E S L A 28H G E G S F S D E L S T I L E A L A 29 H G E G S F S D E L A T I L E S LA 30 H G E G S F S D E L S T I L E A L A 31 H G E G S F S D E L S T I LE A L A 32 H G E G S F S D E L S T I L E S L A 33 H G E G S F S D E L ST I L E S L A 34 H G E G S F S D E L S T I L E S L A 35 H G E G S F S DE L S T I L E A L A 36 H G E G S F S D E L A T I L E S L A 37 H G E G SF S D E L S T I L E S L A 38 H G E G S F S D E L S T I L E S L A 39 H GE G S F S D E L S T I L E A L A 40 H G E G S F S D E L S T I L E S L A41 H G E G S F S D E L S T I L E A L A 42 H G E G S F S D E L A T I L ES L A 43 H G E G S F S D E L S T I L E A L A 44 H G E G S F S D E L A TI L E S L A 45 H G E G S F S D E L A T I L E S L A 46 H G E G S F S D EL G T I L E G L A 47 H G E G S F S D E L K T I L E K L A 48 H G E G S FS D E L E T I L E E L A 49 H G E G S F S D E L D T I L E D L A 50 H G EG S F S D E L H T I L E H L A With K6 51 H G E G S F S D E L A T I L E AL A 52 H G E G S F S D E L L T I L E L L A 53 H G E G S F S D E L F T IL E F L A 54 H G E G S F S D E L V T I L E V L A 55 H G E G S F S D E LI T I L E I L A 56 H G E G S F S D E L L T I L E L L A 57 H G E G S F SD E L I T I L E I L A 58 H G E G S F S D E L V T I L E V L A 59 H G E GS F S D E L F T I L E F L A 60 H G E G S F S D E L A T I L E A L A 61 HG E G S F S D E L L T I L E L L A 62 H G E G S F S D E L I T I L E I L A63 H G E G S F S D E L V T I L E V L A 64 H G E G S F S D E L F T I L EF L A 65 H G E G S F S D E L A T I L E A L A 66 H G E G S F S D E L A TI L E A L A 67 H G E G S F S D E L A T I L E A L A 68 H G E G S F S D EL A T I L E A L A 69 H G E G S F S D E L A T I L E A L A 70 H G E G S FS D E L A T I L E A L A 71 H G E G S F S D E L A T I L E A L A Compound19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 HPI 1 A A D F I A W L I A TK I T D 9 2 A L D F I L W L I L T K I T D 19 3 A F D F I F W L I F T K IT D 14 4 A V D F I V W L I V T K I T D 21 5 A I D F I I W L I I T K I TD 22.5 6 A S D F I S W L I S T K I T D 5.2 7 A S D F I S W L I S T K I TD 6.6 8 A S D F I S W L I S T K I T D 6 9 A S D F I S W L I S T K I T D3.2 10 A G D F I G W L I G T K I T D 2.4 11 A G D F I G W L I G T K I TD 6.4 12 A G D F I G W L I G T K I T D 7.8 13 A G D F I G W L I G T K IT D 7.2 14 A G D F I G W L I G T K I T D 4.4 15 A S D F I S W L I S T KI T D 1.2 16 A A D F I A W L I S T K I T D 6.4 17 A A D F I S W L I A TK I T D 6.4 18 A S D F I A W L I A T K I T D 6.4 19 A A D F I S W L I ST K I T D 3.8 20 A S D F I S W L I A T K I T D 3.8 21 A S D F I A W L IS T K I T D 3.8 22 A S D F I S W L I S T K I T D −4 23 A A D F I A W L IA T K I T D 6.4 24 A A D F I A W L I A T K I T D 6.4 25 A S D F I A W LI A T K I T D 3.8 26 A A D F I A W L I A T K I T D 3.8 27 A A D F I S WL I A T K I T D 3.8 28 A S D F I A W L I A T K I T D 3.8 29 A A D F I AW L I S T K I T D 3.8 30 A A D F I S W L I A T K I T D 3.8 31 A A D F IA W L I S T K I T D 3.8 32 A S D F I S W L I A T K I T D −1.4 33 A S D FI A W L I S T K I T D −1.4 34 A A D F I S W L I S T K I T D −1.4 35 A SD F I S W L I S T K I T D −1.4 36 A S D F I S W L I S T K I T D −1.4 37A S D F I A W L I A T K I T D 1.2 38 A A D F I A W L I S T K I T D 1.239 A A D F I S W L I S T K I T D 1.2 40 A A D F I S W L I A T K I T D1.2 41 A S D F I A W L I S T K I T D 1.2 42 A A D F I S W L I S T K I TD 1.2 43 A S D F I S W L I A T K I T D 1.2 44 A S D F I A W L I S T K IT D 1.2 45 A S D F I S W L I A T K I T D 1.2 46 A G D F I G W L I G T KI T D −2 47 A K D F I K W L I K T K I T D −20 48 A E D F I E W L I E T KI T D −18 49 A D D F I D W L I D T K I T D −18 50 A H D F I H W L I H TK I T D −16 With K6 51 A A D F I A W L I A T K I T D K6 9 52 A L D F I LW L I L T K I T D K6 19 53 A F D F I F W L I F T K I T D K6 14 54 A V DF I V W L I V T K I T D K6 21 55 A I D F I I W L I I T K I T D K6 22.556 A S D F I S W L I S T K I T D K6 5.2 57 A S D F I S W L I S T K I T DK6 6.6 58 A S D F I S W L I S T K I T D K6 6 59 A S D F I S W L I S T KI T D K6 3.2 60 A G D F I G W L I G T K I T D K6 2.4 61 A G D F I G W LI G T K I T D K6 6.4 62 A G D F I G W L I G T K I T D K6 7.8 63 A G D FI G W L I G T K I T D K6 7.2 64 A G D F I G W L I G T K I T D K6 4.4 65A S D F I S W L I S T K I T D K6 1.2 66 A A D F I A W L I S T K I T D K66.4 67 A A D F I S W L I A T K I T D K6 6.4 68 A S D F I A W L I A T K IT D K6 6.4 69 A A D F I S W L I S T K I T D K6 3.8 70 A S D F I S W L IA T K I T D K6 3.8 71 A S D F I A W L I S T K I T D K6 3.8

Example 7 Relative Chemical Stability Testing

The compounds, as listed in Table 3, were dissolved in 0.1 M HCl to anominal concentration of 0.5 mM. The samples were incubated 7 days at40° C. in the dark and then diluted to a nominal concentration of 0.2mg/mL and analysed by RP-HPLC for the recovery of the main peak and byLC-MS for confirmation of the identity by mass of the main peak.

The compounds were analyzed in a nominal concentration of 0.2 mg/mL byRP-HPLC and LC-MS on a Phenomenex Gemini C18 column, 3 μm, 110 Å, 3×150mm with the mobile phase of 0.1% TFA in MQW and in MeCN running stepwisefrom 25% to 48% MeCN over 39 minutes: Flow rate was 0.400 mL/min. Thecolumn oven temperature was 50° C., the auto sampler temperature was 4°C. and the UV detection was measured at 220 nm.

The results for the recovery of purity are listed in Table 3.

TABLE 3 Recovery of purity for ZP Compound. ZP No. Recovery (%) 2242 872263 95 2264 * 2266 73 2267 * 2268 * 2269 39 2270 52 2272 * * Eluted inthe washing phase at analytical RP-HPLC. Repeated data shown in example7 with a modified analytical RP-HPLC method.

Four compounds (ZP2264, ZP2267, ZP2268 and ZP2272) were eluted late inthe washing phase by this analytical method and an experiment wasrepeated with these four compounds by a modified method as described inthe following.

Example 8 Relative Chemical Stability Testing

The compounds, as listed in Table 4, were dissolved in 0.1 M HCl to anominal concentration of 0.25 mM. The samples were incubated 2 days at40° C. in the dark and then diluted to a nominal concentration of 0.2mg/mL and analysed by RP-HPLC for the recovery of the main peak.

The compounds were analyzed in a nominal concentration of 0.2 mg/mL byRP-HPLC and LC-MS on a Phenomenex Gemini C18 column, 3 μm, 110 Å, 3×150mm with the mobile phase 0.1% TFA in MQW and in MeCN running stepwisefrom 25% to 80% MeCN over 39 minutes. Flow rate was 0.400 mL/min. Thecolumn oven temperature was 50° C., the auto sampler temperature was 4°C. and the UV detection was measured at 220 nm.

The results for the recovery of purity are listed in Table 4.

TABLE 4 Recovery of purity for ZP Compound. ZP No. Recovery (%) 1846 952264 61 2267 61 2268 62 2272 61

Example 9 In Vitro Screening of GLP-2 Analogues for Agonism on the GLP-2Receptor Recombinantly Expressed in BHK-21 Cells *)

The screening was done using BHK21 cells transiently transfected withGLP-2 receptor and the CRE-luciferase reporter gene construct.Dose-response curves and EC₅₀ values were determined using sevenconcentrations from 10 fM to 10 nM and triplicate points for eachconcentration. Control peptide was used on all test plates.

*) The test was performed by AMRI at Budapest, Hungary

Materials and Methods

Materials and Cells Used:

Nunc or Greiner Tissue culture flasks

Nunc or Greiner 10 cm TC Petri dishes (Nunc 172931)

Nunc or Greiner 96-well plates for daughter plates (Nunc 167008)

BHK-21(C13) cells

White 96-well plates (PerkinElmer 6005680) Deep-well plates fordilutions (Nunc 278752)

Glasgow MEM (Sigma G5154)

Phenol red-free DMEM (Invitrogen/Gibco 31053-028)

Fetal calf serum (FCS, Invitrogen/Gibco Zealand batch)

Nonessential amino acids (100×) (Invitrogen/Gibco 11140-035)

200 mM Glutamine (100×) (Invitrogen/Gibco 25030-024)

Penicillin/Streptomycin (100×) (Invitrogen/Gibco 15140-122)

Sodium pyruvate (100×) (Invitrogen/Gibco 11360-039)

D-PBS (Invitrogen/Gibco 14190-094)

1× Trypsin-EDTA solution (Invitrogen/Gibco 25300-054)

Opti-MEM (Invitrogen/Gibco 31985-047)

Lipofectamine 2000 (Invitrogen 11668-027)

CRE-luc vector (Zealand batch)

hGLP2R vector (Zealand batch)

10% (w/v) sterile BSA in DMEM (Sigma 05488)

10 mM (200×) IBMX in DMEM (Sigma 15879)

LucLite kit (Perkin Elmer 6016911)

Topseal-A (Perkin Elmer 6005185)

Media and Buffers

Growth Medium for BHK-21(C13) Cells:

Glasgow MEM+2 mM glutamine (1:100)+10% FCS+1% NEAA+1% Pen/Strep+1 mMsodium pyruvate (1:100).

Antibiotics-Free Transfection Medium for BHK-21(C13) Cells:

Glasgow MEM+2 mM glutamine (1:100)+1% NEAA+1 mM sodium pyruvate (1:100)

Post-Transfection Medium for BHK-21(C13) Cells:

Glasgow MEM+2 mM glutamine (1:100)+0.2% FCS+1% NEAA+1% Pen/Strep+1 mMsodium pyruvate (1:100)

Coating Solution:

D-PBS+1% BSA

Peptide Dissolution Buffer:

D-PBS+0.1% BSA

Stimulation Buffer:

Phenol red-free DMEM+2 mM glutamine (1:100)+50 μM IBMX+0.3% BSA

Protocol for Screening of Peptides on Transfected BHK21(C13) Cells

BHK-21 cells are seeded in 10 cm TC-Petri dishes and grown to 70-80%confluency. Subsequently, medium is changed to serum-free transfectionmedium. DNA (7.5 μg hGLP-2 receptor vector and 7.5 μg CRE-luciferasevector) and Lipofectamine 2000 are diluted in OptiMEM, combined, andadded to the cells after 20-30 min. Cells are incubated in the incubatorat 37° C., 5% CO₂ for 4 hr, trypsinized, and reseeded at 35,000 cellsper well in 96-well plates. Cells are incubated overnight in full growthmedium in order to allow expression of the transgenes. All plates usedfor the preparation of diluted GLP-2 analogue solutions are pre-coatedwith 1% BSA in phosphate-buffered saline for 2 hr and dried overnight.GLP-2 analogues are dissolved in PBS/0.1% BSA to 250 μM and diluted intwo steps to 10 μM and 100 nM, respectively, in phenol red- andserum-free medium containing phosphodiesterase inhibitor and 0.3% BSA(stimulation buffer). Thereafter, GLP-2 analogues are serially dilutedin the same stimulation buffer to yield concentrations from 10 nM downto 10 fM. Baseline activity is controlled with stimulation buffer alone.These solutions are pre-warmed to 37° C. for 30 min. Cells are rinsedwith pre-warmed (37° C.) stimulation buffer, and incubated with 100 μlof the pre-warmed peptide dilutions and control for 5 hr at 37° C., 5%CO₂. The LucLite enzyme substrate/lysis buffer is prepared shortlybefore the end of the incubation. 100 μl LucLite substrate are added toeach well, and light emission is counted on a suitable counter (e.g.Wallac VictorII). EC50 values are derived from the raw data by fittingwith a four parameter logistic equation (Microcal Origin 5.0 or GraphPad4).

Results are listed in table 5

TABLE 5 Screening of GLP-2 analogues on GLP-2 receptor-expressing BHK21cells ZP ID 2263 2264 2266 2267 2268 2269 2270 2272 2242 pEC50 10.8411.5 9.253 10.87 11.21 10.04 10.23 11.27 12.41 SEM 0.1929 0.2419 0.22720.3139 0.2239 0.09546 0.1449 0.1668 0.1971 Conclusion: The resultclearly indicates that all the tested compounds are GLP-2 agonists

Example 10 In Vivo Test, Stimulation of Intestinal Growth as Determinedin Male C57BL Mice

The ability of the present compounds (ZP2264, ZP2266, ZP2267, ZP2268,ZP2242, ZP2269, ZP2270, ZP2272 and ZP2242) to selectively stimulatesmall intestinal mass, relative to colon mass was determined in maleC57BL mice. Individual groups (n 6-10) of mice were given 800 nmol/kg ofeach compound, s.c, once daily for three consecutive days. Forcomparison purposes other groups of animals were given either anequimolar dose of [Gly2]GLP-2 (ZP1559) or vehicle (phosphate bufferedsaline, pH 7.4) in the same dosing regimen Twenty-four hours after thelast dose of compound had been given the mice were sacrificed and thesmall intestine (from the pylorus to the cecum) and the colon (intestinedistal to cecum) was emptied and weighed.

To correct for slight difference in body weight (BW), the organ mass ofthe small intestine (SI) and colon were expressed relative to BW. Thenon-selective reference compound [Gly2]GLP-2 has been reported tostimulate gastrointestinal growth in both esophagus, stomach, smallintestine and colon and to evaluate differences in growth patterninduced by compounds, the small intestine-colon sensitivity index ofcompound X was calculated as:

(SI/Colon)_(X)/(SI/Colon)_([Gly2]GLP-2)%

Compounds with a small intestine-colon sensitivity greater than or equalto 1.10 were considered relatively selective for the small intestine.

TABLE 6 List of the small intestine-colon sensitivity index of testcompounds, relative to the reference compound [Gly2]GLP-2. Position 1 23 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 Reference compound[Gly2]GLP-2 H G D G S F S D E M N T I L D N L A A Small intestineselective compounds ZP2264 H G E G S F S D E L A T I L E A L A A ZP2268H G E G S F S D E L A T I L D N L A A Small intes- tine-colon Positionsensitivity 20 21 22 23 24 25 26 27 28 29 30 31 32 33 index Referencecompound [Gly2]GLP-2 R D F I N W L I Q T K I T D OH 1.00 ± 0.02  Smallintestine selective compounds ZP2264 A D F I A W L I A T K I T D NH2 1.15 ± 0.03* ZP2268 R D F I S W L I S T K I T D NH2 1.10 ±0.03* NTindicates “not tested”. *P < 0.05, vs [Gly2]GLP-2

TABLE 7 Effect of the test compounds on small intestine and colon mass,relative to the reference compound [Gly2]GLP-2. Position 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 19 20 21 [Gly2]GLP-2 H G D G S F S D E M NT I L D N L A A R D ZP2263 H G E G S F S D E L S T I L E S L A A S DZP2264 H G E G S F S D E L A T I L E A L A A A D ZP2266 H G E G S F S DE L E T I L E E L A A E D ZP2267 H G E G S F S D E L S T I L D N L A A RD ZP2268 H G E G S F S D E L A T I L D N L A A R D ZP2269 H G E G S F SD E L K T I L D N L A A R D ZP2270 H G E G S F S D E L N T I L D N L A AR D ZP2272 H G E G S F S D E L A T I L D N L A A R D ZP2242 H G E G S FS S E L S T I L D A L A A R D Position SI (% vs. 22 23 24 25 26 27 28 2930 31 32 33 [Gly2]GLP-2 Colon [Gly2]GLP-2 F I N W L I Q T K I T D OH 100± 1  100 ± 2 ZP2263 F I S W L I S T K I T D NH2 NT NT ZP2264 F I A W L IA T K I T D NH2 126 ± 3*  109 ± 3* ZP2266 F I E W L I E T K I T D NH2 95± 1 101 ± 2 ZP2267 F I A W L I A T K I T D NH2 108 ± 2  109 ± 4 ZP2268 FI S W L I S T K I T D NH2 124 ± 3*  112 ± 4* ZP2269 F I E W L I Q T K IT D NH2 97 ± 2  95 ± 3 ZP2270 F I S W L I S T K I T D NH2 NT NT ZP2272 FI S W L 1 A T K I T D NH2 118 ± 2  105 ± 6 ZP2242 F I A W L 1 A T K I TD K OH 111 ± 2   107± 3 NT indicates “not tested”. *P < 0.05, vs[Gly2]GLP-2

Results

The effects of the test compounds according to the invention weredetermined based on the ability of the peptides to selectively increasesmall intestinal mass, relative to colon mass. In FIGS. 1 and 2 relativesmall intestinal mass vs. vehicle controls following the administrationof the reference compound, [Gly2]GLP-2 and ZP2264, ZP2266, ZP2267,ZP2268, ZP2242, ZP2269 and ZP2272 is shown. In FIGS. 3 and 4 the effectof the test compounds on the ratio of small intestine mass to colonmass, vs. that of the reference compound, [Gly2]GLP-2 is shown. Theratio of small intestine mass to colon mass, in the animals given[Gly2]GLP-2 was standardised to 100%. The test compounds that wereselective for the small intestine, based on the calculated smallintestine-colon sensitivity index of, vs. that of the referencecompound, [Gly2]GLP-2, is shown in Table 6. In the animals given[Gly2]GLP-2 the small intestine-colon sensitivity index is standardisedto 1. The effect of the test compounds on small intestine and colonmass, relative to the reference compound [Gly2]GLP-2 is shown in Table7. In the animals given [Gly2]GLP-2 small intestine and colon mass wasstandardised to 100%.

Example 11 In Vivo Test, Stimulation of Intestinal Growth as Determinedin Male C57BL Mice

The ability of further compounds to selectively stimulate smallintestinal mass, relative to colon mass was determined in male C57BLmice. A similar protocol was used to that described above in Example 10except that 200 nmol/kg of each compound was administered per day. Thus,individual groups (n=6-10) of mice were given 200 nmol/kg of eachcompound, s.c, once daily for three consecutive days. Other groups ofanimals were given either an equimolar dose of [Gly2]GLP-2 (referencecompound) or vehicle (phosphate buffered saline, pH 7.4; negativecontrols) in the same dosing regimen. Twenty-four hours after the lastdose of compound the mice were sacrificed and the stomach, smallintestine and the colon emptied and weighed.

As shown in FIG. 6, compounds ZP2380, ZP2381, ZP2384, ZP2385, ZP2397,ZP2398, ZP2399, ZP2411, ZP2417, ZP2418, ZP2420, ZP2423, according to theinvention, have the ability to selectively increase small intestinalmass, relative to colon mass.

Example 12 In Vivo Test, Stimulation of Stomach Growth as Determined inMale C57BL Mice

The effects of the test compounds according to the invention weredetermined based on the ability of the peptides to selectively increasestomach mass, relative to vehicle control. The ability of the presentcompounds (ZP2395, ZP2396, ZP2400, ZP2412, ZP2394 and ZP2401) toselectively stimulate stomach mass, relative to colon mass wasdetermined in male C57BL mice. Individual groups (n=6-10) of mice weregiven 200 nmol/kg of each compound, s.c, once daily for threeconsecutive days. For comparison purposes other groups of animals weregiven either an equimolar dose of [Gly2]GLP-2 or vehicle (phosphatebuffered saline, pH 7.4) in the same dosing regimen. Twenty-four hoursafter the last dose of compound had been given the mice were sacrificedand the stomach and small intestine (from the pylorus to the ileocaecaljunction) emptied and weighed. To correct for slight difference in bodyweight (BW), the organ mass of the stomach is presented relative to BW.In FIG. 7 it is shown that compounds ZP2395, ZP2396, ZP2400, ZP2412,ZP2414, ZP2416, ZP2394 and ZP2401 have the ability to selectivelyincrease stomach mass, relative to colon mass.

The reference compound, [Gly2]GLP-2, has not been reported to stimulatestomach mass, whereas a number of the ZP compounds increased stomachmass relative to bodyweight (FIG. 7). To evaluate the differentialgrowth pattern induced by ZP compounds, compared to [Gly2]GLP-2, on thestomach, the stomach-colon sensitivity index of compound X wascalculated as:

(Stomach/Colon)_(X)/Stomach/(Colon)_([Gly2]GLP-2)%

Compounds with a stomach/colon sensitivity greater than or equal to 1.05were considered relatively selective for the stomach.

The compounds ZP2267, ZP2386, ZP2404, ZP2413, ZP1415, ZP2402, ZP2403,ZP2382, ZP2266, ZP2378, ZP2414, ZP2416, ZP2424, ZP2379, ZP2269 areunselective for the small intestine, colon or stomach (FIG. 8).

Table 8 shows a summary of data obtained in Examples 10 to 12:

TABLE 8 Summary of data Compared to 1559 in % Sensitivity SensitivityColon- index index ZP no. SI-BW BW Sto-BW Si/Colon Stomach/Co 2267 108.5109.4 85.9 0.99 0.79 Non specific peptides 2386 113 111 95 1.02 0.862404 114 115 103 0.99 0.90 2402 110 113 102 0.97 0.90 2413 116 117 1080.99 0.92 2415 106 106 100 1.00 0.94 2403 110 109 107 1.01 0.98 2382 103106 101 0.97 0.95 2266 95.3 100.6 93.6 0.95 0.93 2378 91 97 94 0.94 0.972414 105 106 110 0.99 1.04 2424 86 98 91 0.88 0.93 2416 89 92 96 0.971.04 2379 88 97 99 0.91 1.02 2269 97.4 94.7 99.8 1.03 1.05 2400 106 106111 1.00 1.05 Stomach specific peptides 2412 94 99 106 0.95 1.07 2396104 102 117 1.02 1.15 2395 98 97 113 1.01 1.16 2394 105 96 109 1.09 1.142401 98 88 107 1.11 1.22 2242 111.7 106.6 99.6 1.05 0.93 Small intestinespecific 2411 117 111 106 1.05 0.95 2380 108 102 98 1.06 0.96 2384 128118 100 1.08 0.85 2268 123.6 112.5 87.4 1.10 0.78 2398 108 97 102 1.111.05 2420 112 101 90 1.11 0.89 2417 119 106 90 1.12 0.85 2272 118.1104.7 96 1.13 0.92 2423 123 109 94 1.13 0.86 2264 125.7 109.5 87 1.150.79 2385 110 95 93 1.16 0.98 2399 119 103 115 1.16 1.12 2418 117 100 911.17 0.91 2381 123 104 92 1.18 0.88 2397 122 101 109 1.21 1.08 2263 NTNT NT 2270 NT NT NT 2383 NT NT NT * NT: Not Tested

GLP-2 Receptor Specificity.

The mechanisms by which substrates appear to achieve biologicalspecificity towards a given receptor involves various interactionsbetween the substrate and the receptor based on hydrogen bonding,hydrophobic, electrostatic interactions etc.

During our Structure-Activity Relationship-studies it has appeared thatthe residues in the positions 11, 16, 20, 24 and 28 in the GLP-2sequence are deeply involved in the recognition and binding to the GLP-2receptor. Thus altering the amino acid pattern of these particularpositions divides the GLP-2 analogues into three different groups: smallintestine-, stomach- and non-specific peptides.

The receptor specificity of the GLP-2 analogues was found to depend onthe hydrophobicity and the hydrogen bonding potential of the amino acidsin position 11, 16, 20, 24 and 28.

Example 13 Effect of HPI on Small Intestine Specificity

In table 9 HPI data is shown for the GLP-2 peptides which are smallintestine specific according to the SI/Colon-sensitivity index.

TABLE 9 Small Intestine specific GLP-2 analogues - HydrophaticityProfile (HPP) Compared to 1559 in % Sensitivity Sensitivity ZP SI-Colon- Sto- index index Sequence HPI no. BW BW BW Si/Colon Stomach/Co 1116 20 24 28 11 16 20 24 28 HPP 2242 111.7 106.6 99.6 1.05 0.93 S A R A A−0.8 1.8 −4.5 1.8 1.8 0.1 2411 117 111 106 1.05 0.95 A A A S A 1.8 1.81.8 −0.8 1.8 6.4 2380 108 102 98 1.06 0.96 S S S S S −0.8 −0.8 −0.8 −0.8−0.8 −4 2384 128 118 100 1.08 0.85 A A A A S 1.8 1.8 1.8 1.8 −0.8 6.42268 123.6 112.5 87.4 1.10 0.78 A A S S S 1.8 1.8 −0.8 −0.8 −0.8 1.22398 108 97 102 1.11 1.05 S A S S S −0.8 1.8 −0.8 −0.8 −0.8 −1.4 2420112 101 90 1.11 0.89 S A A A A −0.8 1.8 1.8 1.8 1.8 6.4 2417 119 106 901.12 0.85 A A S A A 1.8 1.8 −0.8 1.8 1.8 6.4 2272 118.1 104.7 96 1.130.92 A S A S A 1.8 −0.8 1.8 −0.8 1.8 3.8 2423 123 109 94 1.13 0.86 A A SA A 1.8 1.8 −0.8 1.8 1.8 6.4 2264 125.7 109.5 87 1.15 0.79 A A A A A 1.81.8 1.8 1.8 1.8 9 2385 110 95 93 1.16 0.98 S A S S S −0.8 1.8 −0.8 −0.8−0.8 −1.4 2399 119 103 115 1.16 1.12 A S S S S 1.8 −0.8 −0.8 −0.8 −0.8−1.4 2418 117 100 91 1.17 0.91 A A A S A 1.8 1.8 1.8 −0.8 1.8 6.4 2381123 104 92 1.18 0.88 A A S S S 1.8 1.8 −0.8 −0.8 −0.8 1.2 2397 122 101109 1.21 1.08 A A A A S 1.8 1.8 1.8 1.8 −0.8 6.4

The data shows that the HPI interval for the individual positions 11,16, 20, 24 and 28 for the small intestine specific GLP-2 analoguesshould independently for position 11 and 16 be at least −0.8≦HPI≦3.8 orpreferably −0.8≦HPI≦2.8 or more preferred HPI=1.8.

For position 20, 24 and 28 the HPI interval should for the individualpositions independently be at least −0.8≦HPI_(20,24,28)≦1.8 orpreferably −0.8≦HPI₂₀≦1.8 and more preferably HPI_(24,28)=−0.8.

Possible amino substitution patterns for small intestine-selectivepeptides according to HPI-intervals are shown in table 10.

TABLE 10 Small Intestine specific GLP-2 analogues - HydrophaticityProfile (HPP) Small intestine specific GLP-2 analogues HPP 11 16 20 2428 HPP preferred A A A A A S S S S S G G G G G T T T T T −4-9 more A A AA A preferred S S S S S G G G T T T −4-9 most A A A A A preferred S S S1.2-9  

Thus each of X11, X16, X20, X24 and X28 may independently be Ala, Ser,Gly or Thr. In particular, each of X11 and X16 may independently be Alaor Ser, and X20, X24 and X28 may independently be Ala, Ser, Gly or Thr.For example, X11 and X16 may both be Ala, and X20, X24 and X28 mayindependently be Ala, Ser, Gly or Thr.

Example 14 Effect of HPI on Stomach Specificity

In table 11 HPI data is shown for the GLP-2 peptides which arestomach-specific according to the Stomach/SI-sensitivity index's.

TABLE 11 Stomach specific GLP-2 analogues - Hydrophaticity Profile (HPP)Compared to 1559 in % Sensitivity Sensitivity ZP SI- Colon- Stomach-index index Sequence HPI no. BW BW BW Si/Co Stomach/Co 11 16 20 24 28 1116 20 24 28 HPP 2400 106 106 111 1.00 1.05 L L S S S 3.8 3.8 −0.8 −0.8−0.8 5.2 2412 94 99 106 0.95 1.07 L L A A A 3.8 3.8 1.8 1.8 1.8 13 2396104 102 117 1.02 1.16 F F S S S 2.8 2.8 −0.8 −0.8 −0.8 3.2 2395 98 97113 1.01 1.16 K K K K K −3.9 −3.9 −3.9 −3.9 −3.9 −19.5 2394 105 96 1091.09 1.14 L L S S S 3.8 3.8 −0.8 −0.8 −0.8 5.2 2401 98 88 107 1.11 1.22F F S S S 2.8 2.8 −0.8 −0.8 −0.8 3.2

The data shows that HPI interval for the individual positions 11, 16,20, 24 and 28 for the stomach specific GLP-2 analogues shouldindependently for the positions 11 and 16 be at least−3.9≦HPI_(11,16)≦3.8 or preferably 2.8≦HPI_(11,16)≦3.8 orHPI_(11,16)=−3.9 and for positions 20, 24 and 28 the HPI shouldindependently be at least −3.9≦HPI_(20,24,28)≦1.8 or preferably−0.8≦HPI_(20,24,28)≦1.8 or HPI_(20,24,28)=−3.9.

Possible amino substitution patterns according to HPI-intervals areshown in table 12.

TABLE 12 Possible amino substitution patterns Stomach specific GLP-2analogues HPI 11 16 20 24 28 HPP Preferred L L A A A F F S S S K K K K13-(−16.4) More L L Preferred F F S S S 5.2-3.2   Thus, forstomach-specific compounds X11 may be Leu, Phe or Lys. X16 may be Leu,Phe or Lys. X20 may be Ala or Ser. X24 may be Ala, Ser or Lys. X28 maybe Ala, Ser or Lys.

For example, X11 and X16 may independently be Leu or Phe, and X20, X24and X28 may be Ser.

Effect of Hydrogen Bonding Potential (HBP) on Receptor Specificity

As mentioned earlier the receptor specificity of the GLP-2 analogues wasalso found to be governed by the hydrogen bonding potential of the abovethe positions 11, 16, 20, 24, and 28.

The hydrogen bonding potential (HBP) was introduced and defined for theindividual amino acids by W. D. Stein, “The movement of molecules acrosscell membranes”; Academic Press N.Y. 1967, pp 65-125, and is providingthe capability of a given amino acid side-chain to make hydrogen bonds.

HBP for the individual amino acids are given in table 13

TABLE 13 HBP for the individual amino acids Amino acid HBP Leu L 0 Ala A0 Cys C 0 Gly G 0 Ile I 0 Met M 0 Phe F 0 Pro P 0 Val V 0 His H 1 Trp W1 Lys K 2 Ser S 2 Thr T 2 Tyr Y 2 Arg R 3 Asn N 3 Asp D 3 Gln Q 3 Glu E3

Example 15 Effect of Hydrogen Bonding Potential (HBP) on StomachSpecificity

Examples of stomach specific analogues are shown in table 14.

TABLE 14 Stomach specific GLP-2 peptide analogues Compared to 1559 in %Sensitivity Sensitivity ZP SI- Colon- Stomach- index index Sequence HBPno. BW BW BW Si/Co Stomach/Co 11 16 20 24 28 11 16 20 24 28 ΣHBP 2400106 106 111 1.00 1.05 L L S S S 0 0 2 2 2 6 2412 94 99 106 0.95 1.07 L LA A A 0 0 0 0 0 0 2396 104 102 117 1.02 1.15 F F S S S 0 0 2 2 2 6 239598 97 113 1.01 1.16 K K K K K 2 2 2 2 2 10 2394 105 96 109 1.09 1.14 L LS S S 0 0 2 2 2 6 2401 98 88 107 1.11 1.22 F F S S S 0 0 2 2 2 6 0-2 0-20-2 0-2 0-2 0-10

According to the table the HBP intervals for the individual positions11, 16, 20, 24 and 28 for the stomach specific GLP-2 analogues shouldindependently for all the positions be at least0≦HBP_(11,16,20,24,28)≦2. In accordance with the HBP intervals, possibleamino acid substitution patterns are shown in table 15.

TABLE 15 Possible amino acid substitution patterns Stomach specificGLP-2 analogues HBP 11  16  20  24  28  Preferred L L A A A F F S S S KK K K HBP 0-2 0-2 0-2 0-2 0-2 More L L K K Preferred F F S S S HBP 0 0 22 2 Most L L S S S preferred F F HBP 0 0 2 2 2 Preferred stomachspecific analogues are GLP-2 analogues with HBP intervals for thepositions 11 and 16 of HBP_(11,16) = 0 and for the positions 20, 24, and28 of independently 0 ≦ HBP_(20,24,28) ≦ 2. Certain stomach specificGLP-2 analogues are as follows: X11 may be Leu, Phe or Lys. X16 may beLeu, Phe or Lys. X20 may be Ala or Ser. X24 may be Ala, Ser or Lys. X28may be Ala, Ser or Lys.

For example, X11 and X16 may independently be Leu or Phe, X24 and X28may independently be Lys or Ser, and X20 may be Ser.

For example, X11 and X16 may independently be Leu or Phe, and X20, X24and X28 may be Ser.

Example 16 Effect of Hydrogen Bonding Potential (HBP) on Small IntestineSpecificity

Small intestine specific GLP-2 analogues was also found to be governedby the parameters such as the hydrogen bonding potential of the aboveparticular mentioned positions 11, 16, 20, 24, and 28. HBP for theindividual amino acids are given in table 16.

TABLE 16 Effect of hydrogen bonding potential (HBP) on small intestinespecificity Compared to 1559 in % Sensitivity Sensitivity ZP SI- Colon-Sto- index index Sequence HBP no. BW BW BW Si/Co Stomach/Co 11 16 20 2428 11 16 20 24 28 ΣHBP 2242 111.7 106.6 99.6 1.05 0.93 S A R A A 2 0 3 00 5 2411 117 111 106 1.05 0.95 A A A S A 0 0 0 2 0 2 2380 108 102 981.06 0.96 S S S S S 2 2 2 2 2 10 2384 128 118 100 1.08 0.85 A A A A S 00 0 0 2 2 2268 123.6 112.5 87.4 1.10 0.78 A A S S S 0 0 2 2 2 6 2398 10897 102 1.11 1.05 S A S S S 2 0 2 2 2 8 2420 112 101 90 1.11 0.89 S A A AA 2 0 0 0 0 2 2417 119 106 90 1.12 0.85 A A S A A 0 0 2 0 0 2 2272 118.1104.7 96 1.13 0.92 A S A S A 0 2 0 2 0 4 2423 123 109 94 1.13 0.86 A A SA A 0 0 2 0 0 2 2264 125.7 109.5 87 1.15 0.79 A A A A A 0 0 0 0 0 0 2385110 95 93 1.16 0.98 S A S S S 2 0 2 2 2 8 2399 119 103 115 1.16 1.12 A SS S S 0 2 2 2 2 8 2418 117 100 91 1.17 0.91 A A A S A 0 0 0 2 0 2 2381123 104 92 1.18 0.88 A A S S S 0 0 2 2 2 6 2397 122 101 109 1.21 1.08 AA A A S 0 0 0 0 2 2 0-2 0-2 0-3 0-2 0-2 0-11

According to the table the HBP intervals for the individual positions11, 16, 20, 24 and 28 for the small intestine specific GLP-2 analoguesshould independently for all the positions be at least0≦HBP_(11,16,20,24,28)≦2.

Preferred analogues have the HBP interval for position 11 and 16HBP_(11,16)=0 and the positions 20, 24, and 28 have independently theHBP interval of 0≦HBP_(20,24,28)≦2.

More preferred GLP-2 analogues showing small intestine specificity arepeptides with an HBP interval for position 11 and 16 of HBP_(11,16)=0,HBP₂₀=0-2, and HBP_(24,28)=2.

Certain substitution patterns are thus shown in Table 17.

TABLE 17 Small intestine specificity peptides Small intestine specificGLP-2 analogues HBP 11 16 20 24 28 preferred A A A A A S S S S S G G G GG T T T T T HBP 0-2 0-2 0-2 0-2 0-2 more A A A A A preferred S S S S S GG G T T T HBP 0-2 0-2 0-2 0-2 0-2 most A A A A A preferred S S S G G G TT T HBP  0  0 0-2 0-2 0-2

Thus, in small intestine-selective compounds, each of X11, X16, X20, X24and X28 may independently be Ala, Ser, Gly or Thr. For example, each ofX11 and X16 may independently be Ala or Ser, and X20, X24 and X28 mayindependently be Ala, Ser, Gly or Thr. For example, X11 and X16 may bothbe Ala, and X20, X24 and X28 may independently be Ala, Ser, Gly or Thr.

While the invention has been described in conjunction with the exemplaryembodiments described above, many equivalent modifications andvariations will be apparent to those skilled in the art when given thisdisclosure. Accordingly, the exemplary embodiments of the invention setforth are considered to be illustrative and not limiting. Variouschanges to the described embodiments may be made without departing fromthe spirit and scope of the invention. All documents cited herein areexpressly incorporated by reference.

1. A glucagon-like peptide 2 (GLP-2) analogue represented by the generalFormula I:R¹—Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-X18-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²wherein: R¹ is hydrogen, C₁₋₄ alkyl, methyl, acetyl, formyl, benzoyl ortrifluoroacetyl; X2 is Gly, Ala or Sar; X3 is Glu or Asp; X5 is Ser orThr; X6 is Phe or Pro or a conservative substitution; X7 is Ser or Thr;X8 is Asp or Ser or a conservative substitution; X9 is Glu or Asp or aconservative substitution; X10 is Met, Leu, Nle or an oxidatively stableMet-replacement amino acid; X11 is Y1; X12 is Thr or Lys or aconservative substitution; X13 is Ile, Glu or Gln or a conservativesubstitution; X14 is Leu, Met or Nle or a conservative substitution; X15is Asp or Glu or a conservative substitution; X16 is Y2; X17 is Leu orGlu or a conservative substitution; X18 is Ala or Aib or anon-conservative substitution; X19 is Ala or Thr or a conservativesubstitution; X20 is Y3 X21 is Asp or Ile or a conservativesubstitution; X24 is Y4; X28 is Y5; X31 is Pro, Ile or deleted; X32 isThr or deleted; X33 is Asp, Asn or deleted; R² is NH₂ or OH; wherein Z¹and Z² are independently absent or a peptide sequence of 1-10 amino acidunits selected from the group consisting of Ala, Leu, Ser, Thr, Tyr,Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn; and the hydrophaticityprofile (HPP) of the residues X11, X16, X20, X24, X28 of formula Icalculated asHPP=Σhpi_(X11)+hpi_(X16)+hpi_(X20)+hpi_(X24)+hpi_(X28) is ≧−10 wherein‘Y₁, Y₂, Y₄, and Y₅ can individually be selected from the groupconsisting of Asn, Asp, Glu, Gln, Lys, His, Arg, Ala, Ser, Thr, Pro,Gly, Leu, Ile, Val, Met or Phe; and Y₃ can be selected from the groupconsisting of Asn, Asp, Glu, Gln, His, Arg, Ala, Ser, Thr, Pro, Gly,Leu, Ile, Val, Met or Phe; with the proviso that when X20 is Arg thenX11 is Ser, X16 is Ala, X24 is Ala, X28 is Ala and Z2 is Lys, or apharmaceutically acceptable salt or derivative thereof.
 2. Aglucagon-like peptide 2 (GLP-2) analogue according to claim 1 whereinHPP is ≧−4.
 3. A glucagon-like peptide 2 (GLP-2) analogue according toclaim 1 wherein HPPI≧0.
 4. A glucagon-like peptide 2 (GLP-2) analoguerepresented by general Formula II:R¹—Z¹-His-X2-X3-Gly-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-X17-Ala-X19-X20-X21-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-X31-X32-X33-Z²—R²wherein: R¹ is hydrogen, C₁₋₄ alkyl, methyl, acetyl, formyl, benzoyl ortrifluoroacetyl X2 is Gly, Ala or Sar X3 is Glu or Asp X5 is Ser or ThrX6 is Phe or Pro X7 is Ser or Thr X8 is Asp or Ser X9 is Glu or Asp X10is Met, Leu, Nle or an oxidatively stable Met-replacement amino acid X11is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val X12 isThr or Lys X13 is Ile, Glu or Gln X14 is Leu, Met or Nle X15 is Asp orGlu X16 is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or ValX17 is Leu or Glu X18 is Ala or Aib X19 is Ala or Thr X20 is Asn, Arg,Ala, Glu, Gly, Ile, Leu, Met, Phe Ser, Thr or Val X21 is Asp or Ile X24is Asn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val X28 isAsn, Ala, Glu, Gly, Ile, Leu, Lys, Met, Phe Ser, Thr or Val X31 is Pro,Ile or deleted X32 is Thr or deleted X33 is Asp, Asn or deleted R² isNH₂ or OH; Z¹ and Z² are independently absent or a peptide sequence of1-10 amino acid units selected from the group consisting of Ala, Leu,Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met and Orn; or apharmaceutically acceptable salt or derivative thereof; with the provisothat when X20 is Arg then X11 is Ser, X16 is Ala, X24 is Ala, X28 is Alaand Z2 is Lys.
 5. A GLP glucagon-like peptide 2 (GLP-2) analogueaccording to claim 4 wherein X11 is Ala, Gly, Ile, Leu, Phe Ser, Thr orVal X16 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val X20 is Ala, Gly, Ile,Leu, Phe Ser, Thr or Val X24 is Ala, Gly, Ile, Leu, Phe Ser, Thr or ValX28 is Ala, Gly, Ile, Leu, Phe Ser, Thr or Val
 6. A GLP glucagon-likepeptide 2 (GLP-2) analogue according to claim 4 wherein X11 is Ala, Ile,Leu, Phe or Val; X16 is Ala, Ile, Leu, Phe or Val X20 is Ala, Ile, Leu,Phe or Val X24 is Ala, Ile, Leu, Phe or Val X28 is Ala, Ile, Leu, Phe orVal
 7. The GLP-2 analogue of claim 1, wherein the GLP-2 analogue has atleast 60% amino acid sequence identity to wild-type GLP-2 (1-33) and hasthe biological activity of causing an increase in intestinal mass invivo.
 8. The GLP-2 analogue of claim 1, wherein the GLP-2 analoguecomprises more than one of the substitutions at positions X11, X16, X20,X24 and/or X28 and/or one of more of said substitutions in combinationwith one or more substitutions at positions X3, X5, X7 and/or X10. 9.The GLP-2 analogue of claim 1, wherein said substitution at position X10is Leu, Nle, or an oxidatively stable Met-replacement amino acid, suchas Met(O) or Met(O)₂.
 10. A GLP-2 analogue is defined represented by thegeneral formula III:R1-His-Gly-Glu-Gly-Ser-Phe-Ser-X8-Glu-Leu-X11-Thr-Ile-Leu-X15-X16-Leu-Ala-Ala-X20-Asp-Phe-Ile-X24-Trp-Leu-Ile-X28-Thr-Lys-Ile-Thr-Asp-NH_(2;)wherein R¹ is hydrogen, C₁₋₄ alkyl, methyl, acetyl, formyl, benzoyl ortrifluoroacetyl X8 is Asp or Ser, preferably Asp; X11 is Ser, Ala, Glu,Lys or Asn; X15 is Glu or Asp, preferably Glu; X16 is Ser, Ala or Glu;X20 is Ser, Ala, or Glu; X24 is Ser, Ala or Glu; and X28 is Ser, Ala,Gln or Glu; or a pharmaceutically acceptable salt or derivative thereof.11. The GLP-2 analogue of claim 1 which is disclosed in Table 1 or Table2, or a pharmaceutically acceptable salt or derivative thereof.
 12. TheGLP-2 analogue of claim 11 which is: 2263HGEGSFSDELSTILESLAASDFISWLISTKITD-NH₂ 2264HGEGSFSDELATILEALAAADFIAWLIATKITD-NH₂ 2266HGEGSFSDELETILEELAAEDFIEWLIETKITD-NH₂ 2267HGEGSFSDELSTILESLAAADFIAWLIATKITD-NH₂ 2268HGEGSFSDELATILEALAASDFISWLISTKITD-NH₂ 2269HGEGSFSDELKTILESLAAADFIEWLIQTKITD-NH₂ 2270HGEGSFSDELNTILESLAASDFISWLISTKITD-NH₂ 2272HGEGSFSDELATILESLAAADFISWLIATKITD-NH₂ 2242HGEGSFSSELSTILDALAARDFIAWLIATKITDK-OH


13. The GLP-2 analogue of claim 1, wherein the GLP-2 analogue comprisesmore than one of the substitutions at positions X3, X5, X7, X11, X16,X20, X24, X28, X31, X32 and/or X33.
 14. The GLP-2 analogue of claim 13,wherein the GLP-2 analogue comprises one or more of substitutionsselected from X11, X16, X20, X24, X28 is Ile, Ala, Leu, Phe or Val; andthe amino acid residues in positions X31, X32 and X33 are optionallydeleted; or a pharmaceutically acceptable salt or derivative thereof.15. The GLP-2 analogue of claim 1 which possesses a substitution at oneor more of positions X11, X16, X20, X24 and/or X28.
 16. The GLP-2analogue of claim 1, wherein each of X11, X16, X20, X24 and X28 isindependently selected from Ala, Ser, Gly and Thr, and wherein theanalogue has preferential growth promoting activity for the smallintestine over the colon.
 17. The GLP-2 analogue of claim 16 whereineach of X11 and X16 is independently selected from Ala and Ser, and X20,X24 and X28 are independently selected from Ala, Ser, Gly and Thr. 18.The GLP-2 analogue of claim 17 wherein X11 and X16 are both Ala, andX20, X24 and X28 are independently selected from Ala, Ser, Gly and Thr.19. The GLP-2 analogue of claim 17 wherein X11 and X16 are both Ala, andX20, X24 and X28 are independently selected from Ala and Ser.
 20. TheGLP-2 analogue of claim 16 comprising one of the following combinationsof residues at positions X11, X16, X20, X24 and X28:Ser/Ser/Ser/Ser/Ser; Ala/Ala/Ser/Ser/Ser, Ala/Ala/Ala/Ala/Ser,Ser/Ala/Ser/Ser/Ser, Ala/Ala/Ala/Ser/Ala; Ala/Ala/Ser/Ala/Ala;Ser/Ala/Ala/Ala/Ala; Ser/Ala/Arg/Ala/Ala; Ala/Ser/Ala/Ser/Ala;Ala/Ala/Ala/Ala/Ala.
 21. The GLP-2 analogue of claim 16 which is ZP2264,ZP2268, ZP2242, ZP2272, ZP2411, ZP2380, ZP2384, ZP2398, ZP2417, ZP2423,ZP2385, ZP2399, ZP2418, ZP2381, ZP2420 or ZP2397.
 22. The GLP-2 analogueof claim 1, wherein X11 is selected from Leu, Phe and Lys; X16 isselected from Leu, Phe and Lys; X20 is selected from Ala, Ser, Leu, Glyand Thr; X24 is selected from Ala and Ser; X28 is selected from Ala, Seror Lys; and wherein the analogue has preferential growth promotingactivity in the stomach compared to colon.
 23. The GLP-2 analogue ofclaim 22 wherein X11 and X16 are independently selected from Leu andPhe, X24 and X28 are independently selected from Lys and Ser, and X20 isSer.
 24. The GLP-2 analogue of claim 23 wherein X11 and X16 areindependently selected from Leu and Phe, and X20, X24 and X28 are Ser.25. The GLP-2 analogue of claim 22 comprising one of the followingcombinations of residues at positions X11, X16, X20, X24 and X28:Lys/Lys/Lys/Lys/Lys; Phe/Phe/Ser/Ser/Ser; Leu/Leu/Ser/Ser/Ser;Leu/Leu/Ala/Ala/Ala.
 26. The GLP-2 analogue of claim 22 which is ZP2400,ZP2412, ZP2396, ZP2394, ZP2401 or ZP2395.
 27. A GLP-2 analogue claim 1wherein the HBP intervals for the individual positions 11, 16, 20, 24and 28 for the small intestine specific GLP-2 analogues shouldindependently for all the positions be at least0≦HBP_(11,16,20,24,28)≦2.
 28. A GLP-2 analogue of claim 27 wherein theHBP interval for position 11 and 16 HBP_(11,16)=0 and the positions 20,24, and 28 have independently the HBP interval of 0≦HBP_(20,24,28)≦2.29. A GLP-2 analogue of claim 1 wherein the HBP intervals for theindividual positions 11, 16, 20, 24 and 28 for the stomach specificGLP-2 analogues should independently for all the positions be at least0≦HBP_(11,16,20,24,28)≦2.
 30. A GLP-2 analogue of claim 29 wherein theHBP intervals for the positions 11 and 16 of HBP_(11,16)=0 and for thepositions 20, 24, and 28 of independently 0≦HBP_(20,24,28)≦2. 31-56.(canceled)